Background In previous work, we investigated dieldrin cytotoxicity and signaling cell death mechanisms in dopaminergic PC12 cells. Dieldrin has been reported to be one of the environmental factors correlated with Parkinson's disease and may selectively destroy dopaminergic neurons. Methods Here we further investigated dieldrin toxicity in a dopaminergic neuronal cell model of Parkinson's disease, namely N27 cells, using biochemical, immunochemical, and flow cytometric analyses. Results In this study, dieldrin-treated N27 cells underwent a rapid and significant increase in reactive oxygen species followed by cytochrome c release into cytosol. The cytosolic cytochrome c activated caspase-dependent apoptotic pathway and the increased caspase-3 activity was observed following a 3 hr dieldrin exposure in a dose-dependent manner. Furthermore, dieldrin caused the caspase-dependent proteolytic cleavage of protein kinase C delta (PKCδ) into 41 kDa catalytic and 38 kDa regulatory subunits in N27 cells as well as in brain slices. PKCδ plays a critical role in executing the apoptotic process in dieldrin-treated dopaminergic neuronal cells because pretreatment with the PKCδ inhibitor rottlerin, or transfection and over-expression of catalytically inactive PKCδK376R, significantly attenuates dieldrin-induced DNA fragmentation and chromatin condensation. Conclusion Together, we conclude that caspase-3-dependentproteolyticactivation of PKCδ is a critical event in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells. PMID:18945348

Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.

Highlights: • AMPK-activation induces caspase3-dependent apoptosis in macrophages. • Apoptosis is associated with decreased mTOR and increased p21 levels. • All effects can be significantly inhibited by the TLR4 agonist lipopolysaccharide. - Abstract: AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner. In conclusion, our results revealed that AMPK activation is not only associated with positive effects but might also contribute to risk factors by disturbing important features of macrophages. The fact that LPS is able to restore AMPK-associated apoptosis might indicate an important role of TLR4 agonists in preventing unfavorable cell death of immune cells.

Axonal injury due to prostatectomy leads to Wallerian degeneration of the cavernous nerve (CN) and erectile dysfunction (ED). Return of potency is dependent on axonal regeneration and reinnervation of the penis. Following CN injury (CNI), RhoA and Rho-associated protein kinase (ROCK) increase in penile endothelial and smooth muscle cells. Previous studies indicate that nerve regeneration is hampered by activation of RhoA/ROCK pathway. We evaluated the role of RhoA/ROCK pathway in CN regulation following CNI using a validated rat model. CNI upregulated gene and protein expression of RhoA/ROCK and caspase-3 mediated apoptosis in the major pelvic ganglion (MPG). ROCK inhibitor (ROCK-I) prevented upregulation of RhoA/ROCK pathway as well as activation of caspase-3 in the MPG. Following CNI, there was decrease in the dimer to monomer ratio of neuronal nitric oxide synthase (nNOS) protein and lowered NOS activity in the MPG, which were prevented by ROCK-I. CNI lowered intracavernous pressure and impaired non-adrenergic non-cholinergic-mediated relaxation in the penis, consistent with ED. ROCK-I maintained the intracavernous pressure and non-adrenergic non-cholinergic-mediated relaxation in the penis following CNI. These results suggest that activation of RhoA/ROCK pathway mediates caspase-3dependent apoptosis of nitrergic neurons in the MPG following CNI and that ROCK-I can prevent post-prostatectomy ED.

Axonal injury due to prostatectomy leads to Wallerian degeneration of the cavernous nerve (CN) and erectile dysfunction (ED). Return of potency is dependent on axonal regeneration and reinnervation of the penis. Following CN injury (CNI), RhoA and Rho-associated protein kinase (ROCK) increase in penile endothelial and smooth muscle cells. Previous studies indicate that nerve regeneration is hampered by activation of RhoA/ROCK pathway. We evaluated the role of RhoA/ROCK pathway in CN regulation following CNI using a validated rat model. CNI upregulated gene and protein expression of RhoA/ROCK and caspase-3 mediated apoptosis in the major pelvic ganglion (MPG). ROCK inhibitor (ROCK-I) prevented upregulation of RhoA/ROCK pathway as well as activation of caspase-3 in the MPG. Following CNI, there was decrease in the dimer to monomer ratio of neuronal nitric oxide synthase (nNOS) protein and lowered NOS activity in the MPG, which were prevented by ROCK-I. CNI lowered intracavernous pressure and impaired non-adrenergic non-cholinergic-mediated relaxation in the penis, consistent with ED. ROCK-I maintained the intracavernous pressure and non-adrenergic non-cholinergic-mediated relaxation in the penis following CNI. These results suggest that activation of RhoA/ROCK pathway mediates caspase-3dependent apoptosis of nitrergic neurons in the MPG following CNI and that ROCK-I can prevent post-prostatectomy ED. PMID:27388816

The folate analogue BGC9331 is a new thymidylate synthase (TS) inhibitor showing a broad spectrum of cyto-toxic activity against several human solid tumors, including colorectal cancer. In this study, we investigated the anticancer activity of BGC9331 either alone or combined with 5-fluorouracil (5-FU), MTA (multi-target antifolate), oxali-platin and SN-38, the active metabolite of the topoisomerase I inhibitor CPT-11. The antiproliferative activity of each drug and BGC9331-based combinations was investigated in the HT-29 human colorectal cancer cell line and its HT-29/5-FU counterparts selected for resistance to 5-FU. BGC9331 combined with MTA or SN-38 induced synergistic responses in HT-29 cells. Treatment of HT-29 cells with either BGC9331 or SN-38 increased caspase-3 activity and the percentage of apoptotic cells from 3 to 13%. Both drugs also augmented the proteolytic cleavage of the Rho-kinase ROCK-1 that was attenuated by the caspase-3 pathway inhibitor z-DEVD-fmk. BGC9331 combined with SN-38 further increased the percentage of apoptotic cells to 25%, and inhibited cell cycle progression and cell proliferation by 65%. This was accompanied by proteolyticactivation of ROCK-1, through both caspase-3-dependent and -independent mechanisms, as shown in caspase-3-deficient MCF-7 breast cancer cells. These encouraging results warrant further preclinical investigations and clinical trials on the use of BGC9331 combined with SN-38/CPT-11 in treatment of patients with advanced colorectal or gastric cancers.

Le Carbone (LC) is a charcoal supplement, which contains a large amount of dietary fibers. Several studies suggested that charcoal supplement may be beneficial for stomach disorders, diarrhea, gas and indigestion. But no studies address whether LC intake would suppress inflammation, cell proliferation or disease progression in colitis. In the present study, the effect of LC on experimental colitis induced by dextran sulfate sodium (DSS) in mice and its possible mechanism of action were examined. A study was designed for 8days, using C57BL/6 female mice that were administered with 3% DSS in drinking water for 7days followed by another 1day consumption of normal water with or without treatment. LC suspension was administered daily for 7days via oral gavage using 5mg/mouse in treatment group and normal group was supplied with drinking water. LC suspension significantly attenuated the loss of body weight and shortening of colon length induced by DSS. The disease activity index, histopathologic changes were significantly reduced by LC treatment. The inflammatory mediators TNFα, IL-1β, p-STAT3 and p-NF-κB induced in the colon by DSS were markedly suppressed by LC. The increased activation of AMPKα in the colon was also detected in LC group. Furthermore, the apoptotic marker protein cleaved caspase 3 was down-regulated and anti-apoptotic proteins Bcl2 and Bcl-xL were significantly up-regulated by LC treatment. Taken together, our results demonstrate the ability of LC to inhibit inflammation, apoptosis and give some evidence for its potential use as adjuvant treatment of inflammatory bowel disease.

In contrast with protein kinase Calpha (PKCalpha) and PKCepsilon, which are better known for promoting cell survival, PKCdelta is known for its pro-apoptotic function, which is exerted mainly through a caspase-3-dependentproteolyticactivation pathway. In the present study, we used the rat GH3B6 pituitary adenoma cell line to show that PKCalpha and PKCepsilon are activated and relocalized together with PKCdelta when apoptosis is induced by a genotoxic stress. Proteolyticactivation is a crucial step used by the three isoforms since: (1) the catalytic domains of the PKCalpha, PKCepsilon or PKCdelta isoforms (CDalpha, CDepsilon and CDdelta respectively) accumulated, and this accumulation was dependent on the activity of both calpain and caspase; and (2) transient expression of CDalpha, CDepsilon or CDdelta sufficed to induce apoptosis. However, following this initial step of proteolyticactivation, the pathways diverge; cytochrome c release and caspase-3 activation are induced by CDepsilon and CDdelta, but not by CDalpha. Another interesting finding of the present study is the proteolysis of PKCdelta induced by CDepsilon expression that revealed the existence of a cross-talk between PKC isoforms during apoptosis. Hence the PKC family may participate in the apoptotic process of pituitary adenoma cells at two levels: downstream of caspase and calpain, and via retro-activation of caspase-3, resulting in the amplification of its own proteolyticactivation. PMID:12238950

The latices of only 13 of a total of 46 species of Ficus examined contained appreciable proteolyticactivity. Therefore, high proteolyticactivity in the latex is not a distinguishing feature of the genus. The latex of F. stenocarpa had the highest specific activity followed closely by the latices of F. carica and F. glabrata. Latices of 6 species of Ficus were examined by chromatography on CM-cellulose and compared with the results obtained for 9 varieties of F. carica. All of the latices were found to contain multiple proteolytic enzymes. Chromatographically, the multiple enzyme components of the several varieties of F. carica were more similar than those of the several species examined. The latices of 16 varieties of F. carica were all different as determined by free boundary electrophoresis although the specific proteolyticactivity of the latices was reasonably constant. PMID:16656886

Placental growth factor (PlGF) is a critical mediator of blood vessel formation, yet mechanisms of its action and regulation are incompletely understood. Here we demonstrate that proteolytic processing regulates the biological activity of PlGF. Specifically, we show that plasmin processing of PlGF-2 yields a protease-resistant core fragment comprising the vascular endothelial growth factor receptor-1 binding site but lacking the carboxyl-terminal domain encoding the heparin-binding domain and an 8-amino acid peptide encoded by exon 7. We have identified plasmin cleavage sites, generated a truncated PlGF118 isoform mimicking plasmin-processed PlGF, and explored its biological function in comparison with that of PlGF-1 and -2. The angiogenic responses induced by the diverse PlGF forms were distinct. Whereas PlGF-2 increased endothelial cell chemotaxis, vascular sprouting, and granulation tissue formation upon skin injury, these activities were abrogated following plasmin digestion. Investigation of PlGF/Neuropilin-1 binding and function suggests a critical role for heparin-binding domain/Neuropilin-1 interaction and its regulation by plasmin processing. Collectively, here we provide new mechanistic insights into the regulation of PlGF-2/Neuropilin-1-mediated tissue vascularization and growth. PMID:23645683

Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway. PMID:27584786

Although information has rapidly developed concerning the intracellular localization of plant proteins, relatively few reports concern the intracellular location of endo- and exo-proteolyticactivities. Relatively few proteases have been purified, characterized, and associated with a specific cellular location. With the exception of the processing proteases involved in transport of proteins across membranes, little progress has yet been made concerning determination of in vivo products of specific proteases. Information on the turnover of individual proteins and the assessment of rate-limiting steps in pathways as proteins are turned over is steadily appearing. Since chloroplasts are the major site of both protein synthesis and, during senescence, degradation, it was important to show unambiguously that chloroplasts can degrade their own constituents. Another important contribution was to obtain evidence that the chloroplasts contain proteases capable of degrading their constituents. This work has been more tenuous because of the low activities found and the possibility of contamination by vacuolar enzymes during the isolation of organelles. The possible targeting of cytoplasmic proteins for degradation by facilitating their transport into vacuoles is a field which hopefully will develop more rapidly in the future. Information on targeting of proteins for degradation via the ubiquitin (Ub) degradation pathway is developing rapidly. Future research must determine how much unity exists across the different eukaryotic systems. At present, it has important implications for protein turnover in plants, since apparently Ub is involved in the degradation of phytochrome. Little information has been developed regarding what triggers increased proteolysis with the onset of senescence, although it appears to involve protein synthesis. Thus far, the evidence indicates that the complement of proteases prior to senescence is sufficient to carry out the observed protein

Although information has rapidly developed concerning the intracellular localization of plant proteins, relatively few reports concern the intracellular location of endo- and exo-proteolyticactivities. Relatively few proteases have been purified, characterized, and associated with a specific cellular location. With the exception of the processing proteases involved in transport of proteins across membranes, little progress has yet been made concerning determination of in vivo products of specific proteases. Information on the turnover of individual proteins and the assessment of rate-limiting steps in pathways as proteins are turned over is steadily appearing. Since chloroplasts are the major site of both protein synthesis and, during senescence, degradation, it was important to show unambiguously that chloroplasts can degrade their own constituents. Another important contribution was to obtain evidence that the chloroplasts contain proteases capable of degrading their constituents. This work has been more tenuous because of the low activities found and the possibility of contamination by vacuolar enzymes during the isolation of organelles. The possible targeting of cytoplasmic proteins for degradation by facilitating their transport into vacuoles is a field which hopefully will develop more rapidly in the future. Information on targeting of proteins for degradation via the ubiquitin (Ub) degradation pathway is developing rapidly. Future research must determine how much unity exists across the different eukaryotic systems. At present, it has important implications for protein turnover in plants, since apparently Ub is involved in the degradation of phytochrome. Little information has been developed regarding what triggers increased proteolysis with the onset of senescence, although it appears to involve protein synthesis. Thus far, the evidence indicates that the complement of proteases prior to senescence is sufficient to carry out the observed protein

Fanconi anemia (FA) is a rare genome instability syndrome with progressive bone marrow failure and cancer susceptibility. FANCJ is one of 17 genes mutated in FA-patients, comprises a DNA helicase that is vital for properly maintaining genomic stability and is known to function in the FA-BRCA DNA repair pathway. While exact role(s) of FANCJ in this repair process is yet to be determined, it is known to interact with primary effector FANCD2. However, FANCJ is not required for FANCD2 activation but is important for its ability to fully respond to DNA damage. In this report, we determined that transient depletion of FANCJ adversely affects stability of FANCD2 and its co-regulator FANCI in multiple cell lines. Loss of FANCJ does not significantly alter cell cycle progression or FANCD2 transcription. However, in the absence of FANCJ, the majority of FANCD2 is degraded by both the proteasome and Caspase-3dependent mechanism. FANCJ is capable of complexing with and stabilizing FANCD2 even in the absence of a functional helicase domain. Furthermore, our data demonstrate that FANCJ is important for FANCD2 stability and proper activation of DNA damage responses to replication blocks induced by hydroxyurea.

The usage of mobile phone increases globally. However, there is still a paucity of data about the impact of electromagnetic fields (EMF) on human health. This study investigated whether EMF radiation would alter the biology of glial cells and act as a tumor-promoting agent. We exposed rat astrocytes and C6 glioma cells to 1950-MHz TD-SCDMA for 12, 24 and 48 h respectively, and found that EMF exposure had differential effects on rat astroctyes and C6 glioma cells. A 48 h of exposure damaged the mitochondria and induced significant apoptosis of astrocytes. Moreover, caspase-3, a hallmark of apoptosis, was highlighted in astrocytes after 48 h of EMF exposure, accompanied by a significantly increased expression of bax and reduced level of bcl-2. The tumorigenicity assays demonstrated that astrocytes did not form tumors in both control and exposure groups. In contrast, the unexposed and exposed C6 glioma cells show no significant differences in both biological feature and tumor formation ability. Therefore, our results implied that exposure to the EMF of 1950-MHz TD-SCDMA may not promote the tumor formation, but continuous exposure damaged the mitochondria of astrocytes and induce apoptosis through a caspase-3-dependent pathway with the involvement of bax and bcl-2. PMID:22870319

Pinguinain is the name given to a proteolytic enzyme preparation obtained from Bromelia pinguin fruits that has been scarcely studied. The present paper deals on the reexamination of the proteases present in fruits of B. pinguin grown in Cienfuegos, Cuba. The preparation (partially purified pinguinain, PPP) showed the main characteristics of the cysteine proteases, i.e., optimum pH within alkaline range (pH 7.2-8.8), inhibition of proteolyticactivity by thiol blocking reagents, which is usually reverted by addition of cysteine, a remarkable thermal stability and notable stability at high ionic strength values. Isoelectric focusing and zymogram of PPP revealed the presence of several proteolytic components between pI 4.6 and 8.1. Preliminary peptidase purification by cationic exchange chromatography showed the presence of two main proteolytic fractions with molecular masses of approximately 20.0 kDa, according to SDS-PAGE.

The Sunn pest, Eurygaster integriceps Puton (Heteroptera: Scutelleridae), is one of the most important pests of wheat and causes considerable damage to this valuable crop annually. Digestive proteinase activity of adult insects was investigated using general and specific substrates and inhibitors. Proteolyticactivity was low when the common conventional substrates, azoalbumin, azocasein and hemoglobin were used to assay salivary glands and midguts. Using the fluorescent casein substrate (BODIPY FL casein), total proteolyticactivity was measured at different pH. Maximum proteolyticactivity was detected at pH 7 (100%) and 8(65%) which suggested the presence of serine proteinases in the salivary glands. There was no detectable proteolyticactivity in midgut extracts. The inhibitors; PMSF (inhibitor of serine proteinases) and TPCK (a specific chymotrypsin inhibitor) showed greater than 50% inhibitory effect on total proteolyticactivity, however, TLCK (specific trypsin inhibitor) and E-64(specific cysteine proteinase inhibitor) did not inhibit total proteolyticactivity. Using fluorescent specific substrates for serine and cysteine proteinases (Z-Arg-AMC, Z-Arg-Arg-AMC, Z-Arg-Phe-AMC and Suc-Ala-Ala-Pro-Phe-AMZ) revealed the presence of tryptic and chymotryptic activity in the salivary gland extract. Zymogram analysis under non-reducing SDS-PAGE conditions and using the substrate APNE showed at least 8 tryptic and chymotryptic activity bands in salivary gland extracts. A single high molecular weight band with tryptic activity (165 kDa) was detected using the substrate BApNA in a zymogram analysis uisng native-PAGE. Kinetic studies showed a km value of 0.6 mM for this enzyme against the substrate BApNA .The inhibitor TLCK decreased activity of the trypsin-like enzyme up to 73% and almost completely eliminated the only band related to this proteinase in the zymogram. Soybean Kunitz type trypsin inhibitor showed no effect on proteolyticactivity of the trypsin

Excessive proteolytic degradation of fibronectin (FN) has been implicated in impaired tissue repair in chronic wounds. We previously reported two strategies for stabilizing FN against proteolytic degradation; the first conjugated polyethylene glycol (PEG) through cysteine residues and the second conjugated PEG chains of varying molecular weight on lysine residues. PEGylation of FN via lysine residues resulted in increased resistance to proteolysis with increasing PEG size, but an overall decrease in biological activity, as characterized by cell and gelatin binding. Our latest method to stabilize FN against proteolysis masks functional regions in the protein during lysine PEGylation. FN is PEGylated while it is bound to gelatin Sepharose beads with 2, 5, and 10 kDa PEG precursors. This results in partially PEGylated FN that is more stable than native FN and whose proteolytic stability increases with PEG molecular weight. Unlike completely PEGylated FN, partially PEGylated FN has cell adhesion, gelatin binding, and matrix assembly responses that are comparable to native FN. This is new evidence of how PEGylation variables can be used to stabilize FN while retaining its activity. The conjugates developed herein can be used to dissect molecular mechanisms mediated by FN stability and functionality, and address the problem of FN degradation in chronic wounds.

The aim of this research was to investigate the potential of previously defined probiotic strain Lactobacillus helveticus M92 as functional starter culture for fermented dairy products. Therefore, proteolyticactivity of L. helveticus M92 was investigated and compared with those of different representatives of probiotic and starter culture strains. Cluster analysis of AFLP fingerprints showed a difference of L. helveticus M92 compared to five other L. helveticus strains, but the percentage of similarity confirmed the identification on species level. Casein hydrolysis by L. helveticus M92 was monitored by agar-well diffusion test, SDS-PAGE and Anson's method. L. helveticus M92 exhibited the highest proteolyticactivity among tested probiotic and starter cultures strains with the fastest acidification rate and the highest pH decrease after overnight incubation in skim milk. The presence of prtH2 gene was confirmed by PCR amplification with specific primers, while PCR product was not obtained after amplification with primers specific to prtH. Furthermore, SDS-PAGE LC-MS/MS analysis of insoluble proteome of L. helveticus M92 enabled identification of several proteins involved in proteolytic system of L. helveticus such as protease PrtM as well as proteins involved in Opp peptide transport system and the intracellular peptidases PepE, PepN, and PepQ.

This communication describes a novel method for detecting of matrix metalloproteinase-7 activity using a peptide substrate labeled with a ferrocene reporter. The substrate serves as a selective ‘electrochemical proteolytic beacon’ (EPB) for this metalloproteinase. The EPB is immobilized on a gold electrode surface to enable ‘on-off’ electrochemical signaling capability for uncleaved and cleaved events. The EPB is efficiently and selectively cleaved by MMP-7 as measured by the rate of decrease in redox current of ferrocene. Direct transduction of a signal corresponding to peptide cleavage events into an electronic signal thus provides a simple, sensitive route for detecting the MMP activity. The new method allows for identification of the activity of MMP-7 in concentrations as low as 3.4 pM. The concept can be extended to design multiple peptide substrate labeled with different electroactive reporters for assaying multiple MMPs activities.

Hyperosmotic shock induces early calpain activation, Smac/DIABLO release from the mitochondria, and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here, we show that JNK1-1 and JNK1-2 are activated early by osmostress, and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated, JNK1-2 is proteolyzed at Asp-385 increasing the release of cytochrome c and caspase-3 activity, thereby creating a positive feedback loop. Expression of Bcl-xL markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-xL co-expression. In these conditions a significant amount of Bid in the cytosol is mono- and bi-ubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp-52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity. PMID:26511318

Hyperosmotic shock induces early calpain activation, Smac/DIABLO release from the mitochondria, and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here, we show that JNK1-1 and JNK1-2 are activated early by osmostress, and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated, JNK1-2 is proteolyzed at Asp-385 increasing the release of cytochrome c and caspase-3 activity, thereby creating a positive feedback loop. Expression of Bcl-xL markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-xL co-expression. In these conditions a significant amount of Bid in the cytosol is mono- and bi-ubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp-52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity.

Antioxidant ellagic acid is a herbal polyphenolic compound shown to possess growth-inhibiting and apoptotic activities in cancer. Protein kinase C (PKC) plays an important role in cell proliferation, apoptosis, and differentiation. Apoptosis of tumor cells is induced by inactivation of glycolytic enzyme of anaerobic metabolism, lactate dehydrogenase (LDH)-A, and by activating apoptotic protein caspase-3 via PKCδ. The present study aims to analyze the role of ellagic acid on regulation of novel and atypical isozymes of PKC to modulate apoptosis and anaerobic metabolism to prevent lymphoma growth as its role on classical PKCs is reported earlier. Expression of novel and atypical isozymes of PKC, activity of PKCδ, expression and activity of caspase-3, and LDH-A have been analyzed. Expression is measured by RT-PCR, activities of PKCδ as level of its catalytic fragment, caspase-3 as level of its p17 fragment, and LDH-A by specific staining. Lymphoma bearing mice were treated with 3 different doses of ellagic acid. The treatment enhanced expression of all novel and atypical PKCs, activity and expression of caspase-3, and activity of PKCδ but decreased activity and expression of LDH-A. Our results suggest that ellagic acid induces apoptosis via novel and atypical PKCs in association with caspase-3 and induces cancer cell death by blocking the energy metabolism.

Polyphenols are natural compounds widely present in fruits and vegetables, which have antimutagenic and anticancer properties. The aim of the present study was to determine the anticancer effect of a polyphenol-rich Aronia melanocarpa juice (AMJ) containing 7.15 g/L of polyphenols in the acute lymphoblastic leukemia Jurkat cell line, and, if so, to clarify the underlying mechanism and to identify the active polyphenols involved. AMJ inhibited cell proliferation, which was associated with cell cycle arrest in G(2)/M phase, and caused the induction of apoptosis. These effects were associated with an upregulation of the expression of tumor suppressor p73 and active caspase 3, and a downregulation of the expression of cyclin B1 and the epigenetic integrator UHRF1. AMJ significantly increased the formation of reactive oxygen species (ROS), decreased the mitochondrial membrane potential and caused the release of cytochrome c into the cytoplasm. Treatment with intracellular ROS scavengers prevented the AMJ-induced apoptosis and upregulation of the expression of p73 and active caspase 3. The fractionation of the AMJ and the use of identified isolated compounds indicated that the anticancer activity was associated predominantly with chlorogenic acids, some cyanidin glycosides, and derivatives of quercetin. AMJ treatment also induced apoptosis of different human lymphoblastic leukemia cells (HSB-2, Molt-4 and CCRF-CEM). In addition, AMJ exerted a strong pro-apoptotic effect in human primary lymphoblastic leukemia cells but not in human normal primary T-lymphocytes. Thus, the present findings indicate that AMJ exhibits strong anticancer activity through a redox-sensitive mechanism in the p53-deficient Jurkat cells and that this effect involves several types of polyphenols. They further suggest that AMJ has chemotherapeutic properties against acute lymphoblastic leukemia by selectively targeting lymphoblast-derived tumor cells.

Morus alba root extract (MARE) has been used to treat hyperglycaemic conditions in oriental medicine. Here, we studied whether MARE possesses a cytotoxic effect on neuroblastoma. To check the cytotoxicity generated by MARE was whether relatively higher against the cancer cells rather than normal cells, we chose a neuroblastoma cell line (B103) and a normal cell line (Rat-2). A CCK assay revealed that MARE (10 μg/ml) reduced cell viability to approximately 60% compared to an untreated control in B103 cells. But in Rat-2 cells, MARE induced relatively lower cytotoxicity. To investigate the mechanisms underlying the cytotoxic effect of MARE, we used flow cytometry combined with immunoblot analyses. We found that MARE-treatment could accumulate ROS and depolarize mitochondria membrane potential of B103 cells. Further treatment with MARE in B103 cells also could damage DNA and induce apoptosis. An expression study of p-Akt also suggested that there was a reduction in cellular proliferation and transcription along with the process of apoptosis, which was further evidenced by an increase in Bax and cleaved-caspase 3 activity. Together, our findings suggest that MARE produces more cytotoxicity in cancer cells while having a relatively attenuated effect on normal cells. As such, MARE may be a safer option in cancer therapeutics, and it also shows potential for the patients with symptoms of hyperglycemia and cancer. PMID:25921607

Temperature and substrate availability constrain the activity of the extracellular enzymes that decompose and release nutrients from soil organic matter (SOM). Proteolytic enzymes are the primary class of enzymes involved in the depolymerization of nitrogen (N) from proteinaceous components of SOM, and their activity affects the rate of N cycling in forest soils. The objectives of this study were to determine whether and how temperature and substrate availability affect the activity of proteolytic enzymes in temperate forest soils, and whether the activity of proteolytic enzymes and other enzymes involved in the acquisition of N (i.e., chitinolytic and ligninolytic enzymes) differs between trees species that form associations with either ectomycorrhizal or arbuscular mycorrhizal fungi. Temperature limitation of proteolytic enzyme activity was observed only early in the growing season when soil temperatures in the field were near 4 degrees C. Substrate limitation to proteolyticactivity persisted well into the growing season. Ligninolytic enzyme activity was higher in soils dominated by ectomycorrhizal associated tree species. In contrast, the activity of proteolytic and chitinolytic enzymes did not differ, but there were differences between mycorrhizal association in the control of roots on enzyme activity. Roots of ectomycorrhizal species but not those of arbuscular mycorrhizal species exerted significant control over proteolytic, chitinolytic, and ligninolytic enzyme activity; the absence of ectomycorrhizal fine roots reduced the activity of all three enzymes. These results suggest that climate warming in the absence of increases in substrate availability may have a modest effect on soil-N cycling, and that global changes that alter belowground carbon allocation by trees are likely to have a larger effect on nitrogen cycling in stands dominated by ectomycorrhizal fungi.

The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is central to lymphocyte activation and lymphomagenesis. MALT1 mediates antigen receptor signalling to NF-κB by acting as a scaffold protein. Furthermore, MALT1 has proteolyticactivity that contributes to optimal NF-κB activation by cleaving the NF-κB inhibitor A20. Whether MALT1 protease activity is involved in other signalling pathways, and the identity of the relevant substrates, is unknown. Here, we show that T-cell receptors (TCR) activation, as well as overexpression of the oncogenic API2-MALT1 fusion protein, results in proteolytic inactivation of CYLD by MALT1, which is specifically required for c-jun N-terminal kinase (JNK) activation and the inducible expression of a subset of genes. These results indicate a novel role for MALT1 proteolyticactivity in TCR-induced JNK activation and reveal CYLD cleavage as the underlying mechanism.

These studies demonstrate that treatment of human U-937 cells with ionizing radiation (IR) is associated with activation of a cytoplasmic myelin basic protein (MBP) kinase. Characterization of the kinase by gel filtration and in-gel kinase assays support activation of a 40 kDa protein. Substrate and inhibitor studies further support the induction of protein kinase C (PKC)-like activity. The results of N-terminal amino acid sequencing of the purified protein demonstrate identity of the kinase with an internal region of PKC delta. Immunoblot analysis was used to confirm proteolytic cleavage of intact 78 kDa PKC delta in control cells to the 40 kDa C-terminal fragment after IR exposure. The finding that both IR-induced proteolyticactivation of PKC delta and endonucleolytic DNA fragmentation are blocked by Bcl-2 and Bcl-xL supports an association with physiological cell death (PCD). Moreover, cleavage of PKC delta occurs adjacent to aspartic acid at a site (QDN) similar to that involved in proteolyticactivation of interleukin-1 beta converting enzyme (ICE). The specific tetrapeptide ICE inhibitor (YVAD) blocked both proteolyticactivation of PKC delta and internucleosomal DNA fragmentation in IR-treated cells. These findings demonstrate that PCD is associated with proteolyticactivation of PKC delta by an ICE-like protease. Images PMID:8557034

The pregnancy-associated glycoproteins (PAGs) represent a complex group of putative aspartic peptidases expressed exclusively in the placentas of species in the Artiodactyla order. The ruminant PAGs segregate into two classes: the 'ancient' and 'modern' PAGs. Some of the modern PAGs possess alterations in the catalytic center that are predicted to preclude their ability to act as peptidases. The ancient ruminant PAGs in contrast are thought to be peptidases, although no proteolyticactivity has been described for these members. The aim of the present study was to investigate (1) if the ancient bovine PAGs (PAG-2 and PAG-12) have proteolyticactivity, and (2) if there are any differences in activity between these two closely related members. Recombinant bovine PAG-2 and PAG-12 were expressed in a baculovirus expression system and the purified proteins were analyzed for proteolyticactivity against a synthetic fluorescent cathepsin D/E substrate. Both proteins exhibited proteolyticactivity with acidic pH optima. The k(cat)/K(m) for bovine PAG-2 was 2.7x10(5) m(-1) s(-1) and for boPAG-12 it was 6.8x10(4) m(-1) s(-1). The enzymes were inhibited by pepstatin A with a K(i) of 0.56 and 7.5 nm for boPAG-2 and boPAG-12, respectively. This is the first report describing proteolyticactivity in PAGs from ruminant ungulates.

The bioinsecticide Bacillus thuringiensis var. israelensis (Bti) is increasingly used worldwide for mosquito control. Although no established resistance to Bti has been described in the field so far, a resistant Aedes aegypti strain (LiTOX strain) was selected in the laboratory using field-collected leaf litter containing Bti toxins. This selected strain exhibits a moderate resistance level to Bti, but a high resistance level to individual Cry toxins. As Bti contains four different toxins, generalist resistance mechanisms affecting mosquito tolerance to different toxins were expected in the resistant strain. In the present work, we show that the resistant strain exhibits an increase of various gut proteolyticactivities including trypsins, leucine-aminopeptidases, and carboxypeptidase A activities. These elevated proteolyticactivities resulted in a faster activation of Cry4Aa protoxins while Cry4Ba or Cry11Aa were not affected. These results suggest that changes in proteolyticactivities may contribute to Bti resistance in mosquitoes together with other mechanisms.

The exuberant expression of proteinases by tumor cells has long been associated with the breakdown of the extracellular matrix, tumor invasion, and metastasis to distant organs. There is both epidemiological and experimental data that support a causative role for proteinases of the matrix metalloproteinase (MMP) family in tumor progression. Optical imaging techniques provide an extraordinary opportunity for non-invasive “molecular imaging” of tumor-associated proteolyticactivity. The application of optical proteolytic beacons for the detection of specific proteinase activities associated with tumors has several potential purposes: 1) Detection of small, early-stage tumors with increased sensitivity due to the catalytic nature of proteolyticactivity, 2) Diagnosis and Prognosis to distinguished tumors that require particularly aggressive therapy or those that will not benefit from therapy, 3) Identification of tumors appropriate for specific anti-proteinase therapeutics and optimization of drug and dose based on determination of target modulation, and 4) as an indicator of efficacy of proteolytically-activated pro-drugs. This chapter describes the synthesis, characterization, and application of reagents that use visible and near infrared fluorescence resonance energy transfer (FRET) fluorophore pairs to detect and measure MMP-referable proteolyticactivity in tumors in mouse models of cancer. PMID:20135290

We studied the effect of melatonin and epithalon on age-related changes in proteolytic digestive enzyme activity in the pancreas and gastric mucosa of rats kept under different lighting conditions. In rats kept under standard illumination, pepsin activity and the total proteolyticactivity in the stomach and pancreas increased by the age of 12 months, but then decreased. Constant and natural lighting disturbed the age dynamics of proteolytic digestive enzyme activity. Administration of melatonin and epithalon to animals exposed to constant lighting restored age dynamics of pepsin activity and little affected total proteolyticactivity.

The main goal of the study was to determine the ability of histones to induce production of the proteolyticallyactive IgG-antibodies in BALB/c mice. In order to perform this study 8 mice were immunized with the fraction of total calf thymus histones. IgGs were isolated from the serum of the immunized and not immunized animals by means of precipitation with 33% ammonium sulfate, followed by affinity chromatography on protein G-Sepharose column. Histones, myelin basic protein (MBP), lysozyme, BSA, ovalbumin, macroglobulin, casein and cytochrome c served as substrates for determining the proteolyticactivity. It was found that IgGs from the blood serum of immunized mice are capable of hydrolyzing histone H1, core histone and MBP. On the contrary, the proteolyticactivity of IgGs from the blood serum of not immunized mice was not detected. The absence of proteolytical enzymes in the fraction of IgGs was proven by HPLC chromatography. High levels of proteolyticactivity toward histones have been also detected in affinity purified IgGs from blood serum of patients with rheumatoid arthritis, but not in healthy donors. These data indicate that eukaryotic histones may induce production of protabzymes in mammals. The possible origin of these protabzymes and their potential biological role in mammalians is discussed.

Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.

The nucleotide sequence of the small nuclear inclusion protein (NIa)-like cistron of plum pox potyvirus (PPV) has been determined. Viral proteolyticactivity was expressed in Escherichia coli cells harboring plasmids with a PPV cDNA insert approximately 7000 nt long. Free PPV capsid protein was detected in these cells, but it was not produced when a mutation was introduced in the PPV cDNA insert which induced a Gln to Pro substitution at the large nuclear inclusion protein (NIb)-capsid protein junction. By mutational analysis, the NIa-like protein was determined to be responsible for the proteolyticactivity. A Gln to Ser substitution at the presumed NIa-NIb junction, which inhibited proteolytic processing at the carboxyl end of the protease, had no effect on proteolytic cleavage at the NIb-capsid protein junction. In contrast with the high efficiency of proteolytic processing at the NIb-capsid protein cleavage site, processing at the ends of the PPV protease was not complete, suggesting that the PPV polyprotein, like that of other potyviruses, contains cleavage sites with different properties.

The objective of this study was to assess the proteolyticactivity of Fasciola hepatica cathepsins in liver sections from mice vaccinated with phage clones of cathepsin L mimotopes, using the film in situ zymography technique. Female BALB/c mice were immunized three times with 2.5 x 10¹¹ phage particles without adjuvant. Animals vaccinated with phage clones produced high titres of anti-mimotope antibodies and a significant reduction in fluke burden was observed following challenge with metacercariae of F. hepatica. The proteolyticactivity in hepatic tissue was reduced after the immunization with phage clones.

Background Proteases are well-known virulence factors that promote survival, pathogenesis and immune evasion of many pathogens. Several lines of evidence suggest that the blood–brain barrier permeability is a prerequisite in microbial invasion of the central nervous system. Because proteases are frequently associated with vascular permeability by targeting junctional proteins, here it is hypothesized that neuropathogenic Escherichia coli K1 exhibit proteolyticactivities to exert its pathogenicity. Methods Zymographic assays were performed using collagen and gelatin as substrates. The lysates of whole E. coli K1 strain E44, or E. coli K-12 strain HB101 were tested for proteolyticactivities. The conditioned media were prepared by incubating bacteria in RPMI-1640 in the presence or absence of serum. The cell-free supernatants were collected and tested for proteases in zymography as mentioned above. Additionally, proteolytic degradation of host immune factors was determined by co-incubating conditioned media with albumin/immunoglobulins using protease assays. Results When collagen or gelatin were used as substrates in zymographic assays, neither whole bacteria nor conditioned media exhibited proteolyticactivities. The conditioned media of neuropathogenic E. coli K1 strain E44, or E. coli K-12 strain HB101 did not affect degradation of albumin and immunoglobulins using protease assays. Conclusions Neither zymographic assays nor protease assays detected proteolyticactivities in either the whole bacteria or conditioned media of E. coli K1 strain E44 and E. coli K-12 strain HB101. These findings suggest that host cell monolayer disruptions and immune evasion strategies are likely independent of proteolyticactivities of neuropathogenic E. coli K1. PMID:23634997

Mouth is an important source of infections and oral infections such as Candida infections increase the risk of mortality. Our purpose was to investigate differences in proteolyticactivity of non-albicans Candida albicans (non-albicans Candida) between clinical isolates and laboratory samples. The second aim was to assess the concentration of pro- and anti-inflammatory cytokine levels IL-1β, IL-10, and TNF-α in saliva of patients with the non-albicans Candida and Candida-negative saliva samples. Clinical yeast samples from our laboratory were used for analyses. Candida strains were grown in YPG at 37 °C for 24 h in water bath with shaking. The activity of Candida proteinases of cell and cell-free fractions were analyzed by MDPF-gelatin zymography. The levels of IL-1β, IL-10, and TNF-α were measured from saliva with ELISA. The study showed differences in the proteolyticactivity among the non-albicans Candida strains. C. tropicalis had higher proteolyticactivity when compared to the other strains. Significant difference was found in salivary IL-1β levels between the non-albicans Candida and control strains (P proteolytic activity among the non-albicans Candida strains. The increased IL-1β concentration may be one of the host response components associated with non-albicans Candida infection.

We examined 62 strains and 21 trade starter cultures from the collection of LB Bulgaricum PLC for proteolytic and antimicrobial activity of lactic acid bacteria (LAB) grown in goat milk. The aim of this study was to investigate the fermentation of caseins, α-lactalbumin and β-lactoglobulin by LAB, using the o-phthaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteolysis targeted mainly caseins, especially β-casein. Whey proteins were proteolyzed, essentially β-lactoglobulin. The proteolyticactivity of Lactococcus lactis l598, Streptococcus thermophilus t3D1, Dt1, Lactobacillus lactis 1043 and L. delbrueckii subsp. bulgaricus b38, b122 and b24 was notably high. The proteolysis process gave rise to medium-sized peptide populations. Most of the examined strains showed antimicrobial activity against some food pathogens, such as Escherichia coli, Staphylococcus aureus, Salmonella cholere enteridis, Listeria monocytogenes, Listeria innocua and Enterobacter aerogenes. The most active producers of antimicrobial-active peptides were strains of L. delbrueckii subsp. bulgaricus and S. thermophilus, which are of practical importance. The starter cultures containing the examined species showed high proteolytic and antimicrobial activity in skimmed goat milk. The greatest antimicrobial activity of the cultures was detected against E. aerogenes. The obtained results demonstrated the significant proteolytic potential of the examined strains in goat milk and their potential for application in the production of dairy products from goat's milk. The present results could be considered as the first data on the proteolytic capacity of strains and starter cultures in goat milk for the purposes of trade interest of LB Bulgaricum PLC.

The aim of this work was to study the proteolyticactivity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA) induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA) in the presence of complete Freund's adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP), bovine serum albumin (BSA), and bovine casein were used as substrates of the proteolyticactivity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolyticactivity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.

Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.

Chromogenic microplate assays in 96 wells were used to determine the stability of enzyme activity in frass of Blattella germanica (Blattodea: Blattellidae). Frass samples were exposed to controlled conditions [temperature 15-35 °C and/or 53-100% relative humidity (RH)] and to household conditions (apartment). Exposure times were 0 (control), 90, 183 and 276 days. Starch digestion and cellulolytic activities decreased during exposure. Non-specific proteolyticactivities were affected by changes in selective proteolyticactivities. Activities towards AAPpNA and SA(3) pNA strongly increased at 100% RH, indicating the possible influence of microorganisms growing on frass. Activities towards BApNA and ArgpNA decreased with increasing decomposition time, whereas activity towards ZRRpNA was not influenced by exposure time. The largest decrease in activities towards ArgpNA and BApNA occurred at temperatures of 15 °C, 30 °C and 35 °C and at 100% RH. Activities towards BApNA and ZRRpNA were very stable under different temperature and RH conditions; this was confirmed by findings showing that these activities were stable in the experimental apartment. In comparison with the control, activities towards ZRRpNA and BApNA after 276 days decreased by 1% and 19%, respectively. The longterm persistence of proteolyticactivities in cockroach frass increases their allergenic hazard potential.

The existing methods for testing proteolyticactivity are time consuming, quite difficult to perform, and do not allow real-time monitoring. Proteases have attracted considerable interest in winemaking and some yeast species naturally present in grape must, such as Metschnikowia pulcherrima, are capable of expressing this activity. In this study, a new test is proposed for measuring proteolyticactivity directly in fermenting grape must, using azocasein, a chromogenic substrate. Several yeast strains were tested and differences in proteolyticactivity were observed. Moreover, analysis of grape must proteins in wines revealed that protease secreted by Metschnikowia strains may be active against wine proteins.

The production of proteolytic enzymes by several strains of ectomycorrhizal fungi i.e., Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), Suillus bovinus (15-3), Suillus luteus (14-7) on mycelia of Trichoderma harzianum, Trichoderma virens and Mucor hiemalis and sodium caseinate, yeast extract was evaluated. The strains of A. muscaria (16-3) and L. laccata (9-12) were characterized by the highest activity of the acidic and neutral proteases. Taking the mycelia of saprotrophic fungi into consideration, the mycelium of M. hiemalis was the best inductor for proteolyticactivity. The examined ectomycorrhizal fungi exhibited higher activity of acidic proteases than neutral ones on the mycelia of saprotrophic fungi, which may imply the participation of acidic proteases in nutrition.

Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]Al) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M[sub r]) 15,000 to 18,000. The authors report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, they found that antibodies to [alpha]Al recognize large (M[sub r] 30,000-35,000) polypeptides as well as typical [alpha]Al processing products (M[sub r] 15,000-18,000). [alpha]Al activity was found in all extracts that had the typical [alpha]Al processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, they made a mutant [alpha]Al in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]Al when the gene is expressed in tobacco. When pro-[alpha]Al was separated from mature [alpha]Al by gel filtration, pro-[alpha]Al was found not to have [alpha]-amylase inhibitory activity. The authors interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. They suggest that the polypeptide cleavage removes a conformation constraint on the precursor to produce the biochemically active molecule. 43 refs., 5 figs., 1 tab.

The latex from the patagonic plant Philibertia gilliesii Hook. et Arn. (Apocynaceae) is a milky-white suspension containing a proteolytic system constituted by several cysteine endopeptidases. A proteolytic preparation (philibertain g) from the latex of P. gilliesii fruits was obtained and characterized to evaluate its potential use in bioprocesses. Philibertain g contained 1.2 g/L protein and a specific (caseinolytic) activity of 7.0 Ucas/mg protein. It reached 80 % of its maximum caseinolytic activity in the pH 7-10 range, retained 80 % of the original activity after 2 h of incubation at temperatures ranging from 25 to 45 °C and could be fully inactivated after 5 min at 75 °C. Philibertain g retained 60 % of the initial activity even at 1 M NaCl and was able to hydrolyze proteins from stickwater one, of the main waste effluents generated during fishmeal production. Furthermore, as a contribution to the knowledge of the proteolytic system of P. gilliesii, we are reporting the purification of a new peptidase, named philibertain g II (pI 9.4, molecular mass 23,977 Da, N-terminus LPESVDWREKGVVFPXRNQ) isolated from philibertain g through a purification scheme including acetone fractionation, cation exchange, molecular exclusion chromatography, and ultrafiltration.

Proteolysis has many important roles in physiological regulation. It is involved in numerous cell signaling processes and the pathogenesis of many diseases, including cancers. Methods of visualizing and assaying proteolyticactivity are therefore in demand. Förster resonance energy transfer (FRET) probes offer several advantages in this respect. FRET supports end-point or real-time measurements, does not require washing or separation steps, and can be implemented in various assay or imaging formats. In this chapter, we describe methodology for preparing self-assembled concentric FRET (cFRET) probes for multiplexed tracking and imaging of proteolyticactivity. The cFRET probe comprises a green-emitting semiconductor quantum dot (QD) conjugated with multiple copies of two different peptide substrates for two target proteases. The peptide substrates are labeled with different fluorescent dyes, Alexa Fluor 555 and Alexa Fluor 647, and FRET occurs between the QD and both dyes, as well as between the two dyes. This design enables a single QD probe to track the activity of two proteases simultaneously. Fundamental cFRET theory is presented, and procedures for using the cFRET probe for quantitative measurement of the activity of two model proteases are given, including calibration, fluorescence plate reader or microscope imaging assays, and data analysis. Sufficient detail is provided for other researchers to adapt this method to their specific requirements and proteolytic systems of interest.

Summary The ubiquitin-proteasome system (UPS) includes 3 enzymes that conjugate ubiquitin to intracellular proteins that are then recognized and degraded in the proteasome. The process participates in the regulation of cell metabolism. In the kidney, the UPS regulates the turnover of transporters and signaling proteins and its activity is down regulated in acidosis-induced proximal tubular cell hypertrophy. In chronic kidney disease (CKD), muscle wasting occurs because complications of CKD including acidosis, insulin resistance, inflammation, and increased angiotensin II levels stimulate the UPS to degrade muscle proteins. This response also includes caspase-3 and calpains which act to cleave muscle proteins to provide substrates for the UPS. For example, caspase-3 degrades actomyosin, leaving a 14kD fragment of actin in muscle. The 14 kD actin fragment is increased in muscle of patient with kidney disease, burn injury and surgery. In addition, acidosis, insulin resistance, inflammation and angiotensin II stimulate glucocorticoid production. Glucocorticoids are also required for the muscle wasting that occurs in CKD. Thus, the UPS is involved in regulating kidney function and participates in highly organized responses that degrade muscle protein in response to loss of kidney function. PMID:18723090

Proteases have a significant role in the life cycle of parasites and the pathogen-host relationship, being regarded as important virulence factors. In the parasitic nematode Hysterothylacium aduncum proteolyticactivity was measured during in vitro development from third larval stage (L3) to mature adult, using DQ red casein as a fluorogenic substrate. Proteolyticactivity was detected in all the developmental stages studied and at all pH values within the range employed (2.0-7.5). The assay with specific inhibitors permitted the determination of metalloprotease activity, and, to a lesser extent, that of aspartate- and cysteine-protease. Serine-protease activity was the lowest of those studied. In L3 recently collected from the host fish (L3-0 h), the greatest activity was found at an optimum pH of 4.0 and was mainly inhibited by 1,10-phenathroline (metalloprotease inhibitor). This metalloprotease activity in L3-0 h (infective stage) may be related to the invasion of the host tissues by this larva. In the other developmental stages, the greatest protease activity was found at pH 5.5, although at pH 4.0 a lower activity peak was detected. On the other hand, our data show that the proteolyticactivity of the nematode varies according to the presence of pepsin (an aspartic-protease) in the culture medium. Thus, at pH 4.0, activity was greater in the absence of pepsin, with increasing aspartic-protease activity. Together with the detection of aspartic-, cysteine- and metallo-protease (enzymes involved in digestion in invertebrates) in all the developmental stages of the parasite taking place in the digestive tract of the host fish, this allows us to suggest that the pepsin in the culture medium mimics the predigestion conditions in the habitat of the worm within the host and that the activity detected may have, amongst others, a digestive function.

Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolyticactivity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolyticactivity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis-Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD-thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.

Protease secretion in Saccharomyces cerevisiae cultures is a complex process, important for the application of this organism in the food industry and biotechnology. Previous studies provide rather quantitative data, yielding no information about the number of enzymes involved in proteolysis and their individual biochemical properties. Here we demonstrate that W303a and BY4742 S. cerevisiae strains reveal different patterns of spontaneous and gelatin-induced extracellular proteolyticactivity. We applied the gelatin zymography assay to track changes of the proteolytic profile in time, finding the protease secretion dependent on the growth phase and the presence of the protein inducer. Detected enzymes were characterized regarding their substrate specificity, pH tolerance, and susceptibility to inhibitors. In case of the W303a strain, only one type of gelatin-degrading secretory protease (presumably metalloproteinase) was observed. However, the BY4742 strain secreted different proteases of the various catalytic types, depending on the substrate availability. Our study brings the evidence that S. cerevisiae strains secrete several kinds of proteases depending on the presence and type of the substrate. Protein induction may cause not only quantitative but also qualitative changes in the extracellular proteolytic patterns.

ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolyticactivity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolyticactivity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolyticactivity in leukemic

The 2,6,9-trisubstituted purine group of cyclin dependent kinase inhibitors have the potential to be clinically relevant inhibitors of cancer cell proliferation. We have recently designed and synthesized a novel dansylated analog of purvalanol B, termed VMY-1-103, that inhibited cell cycle progression in breast cancer cell lines more effectively than did purvalanol B and allowed for uptake analyses by fluorescence microscopy. ErbB-2 plays an important role in the regulation of signal transduction cascades in a number of epithelial tumors, including prostate cancer (PCa). Our previous studies demonstrated that transgenic expression of activated ErbB-2 in the mouse prostate initiated PCa and either the overexpression of ErbB-2 or the addition of the ErbB-2/ErbB-3 ligand, heregulin (HRG), induced cell cycle progression in the androgen-responsive prostate cancer cell line, LNCaP. In the present study, we tested the efficacy of VMY-1-103 in inhibiting HRG-induced cell proliferation in LNCaP prostate cancer cells. At concentrations as low as 1 μM, VMY-1-103 increased both the proportion of cells in G(1) and p21(CIP1) protein levels. At higher concentrations (5 μM or 10 μM), VMY-1-103 induced apoptosis via decreased mitochondrial membrane polarity and induction of p53 phosphorylation, caspase-3 activity and PARP cleavage. Treatment with 10 μM Purvalanol B failed to either influence proliferation or induce apoptosis. Our results demonstrate that VMY-1-103 was more effective in inducing apoptosis in PCa cells than its parent compound, purvalanol B, and support the testing of VMY-1-103 as a potential small molecule inhibitor of prostate cancer in vivo.

Cardiovascular diseases are chronic inflammatory diseases that affect a large segment of society. Coronary heart disease (CHD), the most common cardiovascular disease, progresses over several years and affects millions of people worldwide. Chronic infections may contribute to the systemic inflammation and enhance the risk for CHD. Periodontitis is one of the most common chronic infections that affects up to 50% of the adult population. Under inflammatory conditions the activation of endogenous degradation pathways mediated by immune responses leads to the release of destructive cellular molecules from both resident and immigrant cells. Matrix metalloproteinases (MMPs) and their regulators can activate each other and play an important role in immune response via degrading extracellular matrix components and modulating cytokines and chemokines. The action of MMPs is required for immigrant cell recruitment at the site of inflammation. Stimulated neutrophils represent the major pathogen-fighting immune cells that upregulate expression of several proteinases and oxidative enzymes, which can degrade extracellular matrix components (e.g. MMP-8, MMP-9 and neutrophil elastase). The activity of MMPs is regulated by endogenous inhibitors and/or candidate MMPs (e.g. MMP-7). The balance between MMPs and their inhibitors is thought to mirror the proteolytic burden. Thus, neutrophil-derived biomarkers, including myeloperoxidase, may activateproteolytic destructive cascades that are involved in subsequent immune-pathological events associated with both periodontitis and CHD. Here, we review the existing studies on the contribution of MMPs and their regulators to the infection-related pathology. Also, we discuss the possible proteolytic involvement and role of neutrophil-derived enzymes as an etiological link between chronic periodontitis and CHD.

Rock squirrels (Spermophilus variegatus) from two sites in south central New Mexico, where prairie (Crotalus viridis viridis) and western diamondback (Crotalus atrox) rattlesnakes are common predators, were assayed for inhibition of rattlesnake venom digestive and hemostatic activities. At statistically significant levels rock squirrel blood sera reduced the metalloprotease and hemolytic activity of venoms from C. v. viridis and C. atrox more than venom from an allopatric snake species, the northern Pacific rattlesnake (Crotalus oreganus). In contrast, general proteolyticactivity of venom from C. oreganus was inhibited more by S. variegatus serum defenses than activity of venom from sympatric snakes. For all three venoms, incubation with squirrel sera increased the level of fibrinolysis over venom-only treatments. These results suggest that rock squirrels (S. variegatus) can defend against metalloproteases and other proteases after envenomation from at least two of five rattlesnake predators they might encounter. However, there were statistically significant differences between general proteolyticactivity and fibrinolytic activity of C. v. viridis and C. atrox venom, suggesting that rock squirrels might be differentially vulnerable to these two predators. The hypothesis that prey resistance influences snake venom evolution in a predator-prey arms race is given further support by the previously cryptic variation in venoms detected when assayed against prey defenses.

Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolyticactivity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolyticactivity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications.

Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolyticactivity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolyticactivity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications. PMID:24895632

When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolyticactivity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.

Treatment of the catalytic (C) trimer of Escherichia coli aspartate transcarbamoylase (ATCase) with alpha-chymotrypsin by a procedure similar to that used by Chan and Enns (1978, Can. J. Biochem. 56, 654-658) has been shown to yield an intact, active, proteolytically cleaved trimer containing polypeptide fragments of 26,000 and 8,000 MW. Vmax of the proteolytically cleaved trimer (CPC) is 75% that of the wild-type C trimer, whereas Km for aspartate and Kd for the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, are increased about 7- and 15-fold, respectively. CPC trimer is very stable to heat denaturation as shown by differential scanning microcalorimetry. Amino-terminal sequence analyses as well as results from electrospray ionization mass spectrometry indicate that the limited chymotryptic digestion involves the rupture of only a single peptide bond leading to the production of two fragments corresponding to residues 1-240 and 241-310. This cleavage site involving the bond between Tyr 240 and Ala 241 is in a surface loop known to be involved in intersubunit contacts between the upper and lower C trimers in ATCase when it is in the T conformation. Reconstituted holoenzyme comprising two CPC trimers and three wild-type regulatory (R) dimers was shown by enzyme assays to be devoid of the homotropic and heterotropic allosteric properties characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrate that the holoenzyme reconstituted from CPC trimers is in the R conformation. These results indicate that the intact flexible loop containing Tyr 240 is essential for stabilizing the T conformation of ATCase. Following denaturation of the CPC trimer in 4.7 M urea and dilution of the solution, the separate proteolytic fragments re-associate to form active trimers in about 60% yield. How this refolding of the fragments, docking, and association to form trimers are achieved is not known. PMID:8318885

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolyticactivities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolyticactivity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes. PMID:26273250

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolyticactivities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolyticactivity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

Immunofluorescence and other methods have been used to probe the self-assembly and internalization of the binary toxin, anthrax lethal toxin (LeTx), in primary murine macrophages. Proteolyticactivation of protective antigen (PA; 83 kDa, the B moiety of the toxin) by furin was the rate-limiting step in internalization of LeTx and promoted clearance of PA from the cell surface. A furin-resistant form of PA remained at the cell surface for at least 90 min. Oligomerization of receptor-bound PA63, the 63 kDa active fragment of PA, was manifested by its conversion to a pronase-resistant state, characteristic of the heptameric prepore form in solution. That oligomerization of PA63 triggers toxin internalization is supported by the observation that PA20, the complementary 20 kDa fragment of PA, inhibited clearance of nicked PA. The PA63 prepore, with or without lethal factor (LF), cleared slowly from the cell surface. These studies show that proteolytic cleavage of PA, in addition to permitting oligomerization and LF binding, also promotes internalization of the protein. The relatively long period of activation and internalization of PA at the cell surface may reflect adaptation of this binary toxin that maximizes self-assembly.

The aim of this work was to study the recovery of proteolytic and collagenolytic activities from rayfish (Raja clavata) viscera wastes. Initially, different parts of the gastrointestinal tract by-products (stomach, duodenum section including pancreas, final intestine) were evaluated. The extracts from proximal intestine yielded the highest values of both enzymatic activities. Optimal conditions for protease activity quantification were established at pH = 6, T = 40 degrees C and incubation time < or =20 min. The mathematical equation used to model the joint effect of pH and temperature led to maximum activity at pH = 8.66 and 59.4 degrees C, respectively. Overcooled acetone was found to be best option for recovery of enzymatic activities in comparison with ethanol, PEG-4000, ammonium sulphate and ultrafiltration system. Finally, a simple and systematic protocol of partial purification and total recovery of proteases and collagenases was defined.

The aim of this work was to study the recovery of proteolytic and collagenolytic activities from rayfish (Raja clavata) viscera wastes. Initially, different parts of the gastrointestinal tract by-products (stomach, duodenum section including pancreas, final intestine) were evaluated. The extracts from proximal intestine yielded the highest values of both enzymatic activities. Optimal conditions for protease activity quantification were established at pH = 6, T = 40 °C and incubation time ≤20 min. The mathematical equation used to model the joint effect of pH and temperature led to maximum activity at pH = 8.66 and 59.4 °C, respectively. Overcooled acetone was found to be best option for recovery of enzymatic activities in comparison with ethanol, PEG-4000, ammonium sulphate and ultrafiltration system. Finally, a simple and systematic protocol of partial purification and total recovery of proteases and collagenases was defined. PMID:20098611

The role of micro-organisms on the ripening process of dry-cured ham, particularly with respect to proteolysis, is not clear. This is partially due to the lack of an adequate system to study changes on a sterile control meat product for long ripening times. Using a meat system based on sterile pork loins ripened under aseptic conditions for 106 days, the contribution to the proteolysis of two micro-organisms isolated from dry-cured ham has been established. Changes were studied by SDS-PAGE of sarcoplasmic and myofibrillar proteins, capillary zone electrophoresis (CZE) of low ionic strength-soluble nitrogen compounds, and HPLC of free amino acids. Debaryomyces hansenii Dh345 did not show any significant proteolyticactivity. However, Penicillium chrysogenum Pg222 showed high proteolyticactivity on myofibrillar proteins resulting in an increase in soluble nitrogen compounds. For this, P. chrysogenum Pg222 should be considered to be used as starter culture in meat products made using long ripening times.

With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faecalis (Ent. faecalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (αS1-, αS2-, and β-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Ent. faecalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42°C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecalis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins.

Summary Ligands stimulate Notch receptors by inducing regulated intramembrane proteolysis (RIP) to produce a transcriptional effector. Notch activation requires unmasking of a metalloprotease cleavage site remote from the site of ligand binding, raising the question of how proteolytic sensitivity is achieved. Here, we show that application of physiologically relevant forces to the regulatory switch results in sensitivity to metalloprotease cleavage, and that bound ligands induce Notch signal transduction in cells only in the presence of applied mechanical force. Synthetic receptor-ligand systems that remove the native ligand-receptor interaction also activate Notch by inducing proteolysis of the regulatory switch. Together, these studies show that mechanical force exerted by signal-sending cells is required for ligand-induced Notch activation, and establish that force-induced proteolysis can act as a mechanism of cellular mechanotransduction. PMID:26051539

Human plasma and serum proteins are subject to intrinsic proteolytic degradation both during and after blood collection. By monitoring peptides, we investigated the stability of plasma and serum samples and the effects of anticoagulants and protease inhibitors on the plasma samples. Serum and plasma were subjected to time-course incubation, and the peptides (750-3200 Da) were extracted and analyzed with MALDI-TOF MS. Peptides of interest were further identified by MALDI-TOF/TOF MS and ESI-MS/MS analyses. Our observations indicate that plasma peptides are significantly different from serum peptides. Intrinsic proteases cause these differences between plasma and serum samples, as well as the differences among three plasma samples using either EDTA, sodium citrate, or heparin as the anticoagulant, which accounts for partial inhibitory effects on plasma proteolyticactivities. Proteases and peptidases, including both aminopeptidases and carboxypeptidases, also cause time-dependent, sequential generation and digestion of the peptides in serum and all three plasmas, specifically during early sample collection and processing. Protease inhibitors within an EDTA-plasma-collection device inhibit both intrinsic plasma peptidases and proteases and moderate the time-dependent changes of the plasma peptides, including bradykinin, and complement C4- and C3- derived peptides. Our results suggest that mixing protease inhibitors immediately with blood during blood collection provides enhanced stabilization of the plasma proteome.

Circulating proteolyticactivity (PA) increases following burn or surgical trauma. Challenging traumatized mice with the yeast Candida albicans further increases PA. Once a PA threshold has been passed, mortality increases as PA increases. The purposes of this study were to determine (i) if gram-negative bacterial challenge affects circulating PA and mortality as Candida challenge does and (ii) if proteinase inhibitor treatment with aprotinin, antithrombin III, and alpha 1-proteinase inhibitor decreases circulating PA and increases the survival of burned mice infected with a bacterium. For all bacteria tested (Proteus mirabilis, Pseudomonas aeruginosa, and Klebsiella pneumoniae), burn plus challenge significantly elevated PA and mortality above levels in mice that were only burned or only challenged. Quantitative culture counts indicated that the mice died of sepsis. Proteinase inhibitor treatment of mice burned and challenged with K. pneumoniae significantly decreased circulating PA, decreased the hepatic microbial load, and increased survival. Hence, in traumatized mice challenged with either C. albicans or gram-negative bacteria, a relationship exists between proteolytic load and subsequent septic death. Parallels between these animal studies and human studies are discussed. PMID:8188336

Thirty strains of Penicillium roqueforti used for the production of blue cheeses were examined for proteolyticactivity by agar diffusion on casein agar, by the azocasein test and by capillary zone electrophoresis (CE). Distinct differences were seen between the strains by all three methods applied and the 30 strains could be subdivided in three groups being significantly different in their proteolyticactivity as measured by the agar diffusion test. The quantitative differences seen in the agar diffusion test were confirmed by the azocasein test. However, a negative result on casein agar, i.e., no clearing of the agar was observed for one strain while it showed low proteolyticactivity in the azocasein test. CE proved to be a valuable method for revealing qualitative differences between strains of P. roqueforti in the breakdown of casein. Three strongly proteolytic strains broke down the specific casein fractions differently: one strain broke down betaA1-casein faster than betaA2-casein, the second preferred alpha s1-casein while the last strain broke down the casein fractions at equal rates. For a strain with medium proteolyticactivity, the degradation of casein was seen by the appearance of a peak with migration time similar to alpha s1-I casein, a peptide normally produced by chymosin.

The study of the basal (BAP) and stimulated (SAP) acid production effect on the average daily acidity and speed of proteolysis in the stomach in vivo was conducted in 498 patients with various functional states of the stomach. It was established that the aforesaid muriatic secretion indices influence the acidity and speed of proteolysis in natural conditions of digestion only within the low and normal range of their values. In the condition of HCl hypersecretion, the dependence between the muriatic secretion and acidity and proteolytic stomach activity in vivo gets lost. This can be explained by the autoregulation effect supporting the introgastral pH optimal for pepsin activity irrespective of the intensity of the acid glands hyperplasia.

It had been shown previously that the heat-stable polypeptide of the ATP-dependent proteolytic system of reticulocytes, designated APF-1, forms covalent conjugates with protein substrates in an ATP-requiring process. We now describe an enzyme that carries out the activation by ATP of the polypeptide with pyrophosphate displacement. The formation of AMP-polypeptide and transfer of the polypeptide to a secondary acceptor are suggested by an APF-1 requirement for ATP-PPi and ATP-AMP exchange reactions, respectively. With radiolabeled polypeptide, an ATP-dependent labeling of the enzyme was shown to be by a linkage that is acid stable but is labile to treatment with mild alkali, hydroxylamine, borohydride, or mercuric salts. It therefore appears that the AMP-polypeptide undergoes attack by an -SH group of the enzyme to form a thiolester. PMID:6262770

MALT1 is a signaling protein that plays a key role in immunity, inflammation, and lymphoid malignancies. For a long time MALT1 was believed to function as a scaffold protein, providing an assembly platform for other signaling proteins. This view changed dramatically when MALT1 was also found to have proteolyticactivity and a capacity to fine-tune immune responses. Preclinical studies have fostered the belief that MALT1 is a promising therapeutic target in autoimmunity and B cell lymphomas. However, recent studies have shown that mice expressing catalytically-inactive MALT1 develop multi-organ inflammation and autoimmunity, and thus have tempered this initial enthusiasm. We discuss recent findings, highlighting the urgent need for a better mechanistic and functional understanding of MALT1 in host defense and disease.

NUB1 (Nedd8 ultimate buster 1) is an adaptor protein which negatively regulates the ubiquitin-like protein Nedd8 as well as neddylated proteins levels through proteasomal degradation. However, molecular mechanisms underlying this function are not completely understood. Here, we report that the oncogenic E3 ubiquitin ligase Mdm2 is a new NUB1 interacting protein which induces its ubiquitination. Interestingly, we found that Mdm2-mediated ubiquitination of NUB1 is not a proteolytic signal. Instead of promoting the conjugation of polyubiquitin chains and the subsequent proteasomal degradation of NUB1, Mdm2 rather induces its di-ubiquitination on lysine 159. Importantly, mutation of lysine 159 into arginine inhibits NUB1 activity by impairing its negative regulation of Nedd8 and of neddylated proteins. We conclude that Mdm2 acts as a positive regulator of NUB1 function, by modulating NUB1 ubiquitination on lysine 159. PMID:28099510

Tolerance of Aspergillus nidulans to alkalinity and elevated cation concentrations requires both SltA and SltB. Transcription factor SltA and the putative pseudokinase/protease signaling protein SltB comprise a regulatory pathway specific to filamentous fungi. In vivo, SltB is proteolytically cleaved into its two principal domains. Mutational analysis defines a chymotrypsin-like serine protease domain that mediates SltB autoproteolysis and proteolytic cleavage of SltA. The pseudokinase domain might modulate the protease activity of SltB. Three forms of the SltA transcription factor coexist in cells: a full-length, 78-kDa version and a processed, 32-kDa form, which is found in phosphorylated and unphosphorylated states. The SltA32kDa version mediates transcriptional regulation of sltB and, putatively, genes required for tolerance to cation stress and alkalinity. The full-length form, SltA78kDa, apparently has no transcriptional function. In the absence of SltB, only the primary product of SltA is detectable, and its level equals that of SltA78kDa. Mutations in sltB selected as suppressors of null vps alleles and resulting in cation/alkalinity sensitivity either reduced or eliminated SltA proteolysis. There is no evidence for cation or alkalinity regulation of SltB cleavage, but activation of sltB expression requires SltA. This work identifies the molecular mechanisms governing the Slt pathway. PMID:27307585

Tolerance of Aspergillus nidulans to alkalinity and elevated cation concentrations requires both SltA and SltB. Transcription factor SltA and the putative pseudokinase/protease signaling protein SltB comprise a regulatory pathway specific to filamentous fungi. In vivo, SltB is proteolytically cleaved into its two principal domains. Mutational analysis defines a chymotrypsin-like serine protease domain that mediates SltB autoproteolysis and proteolytic cleavage of SltA. The pseudokinase domain might modulate the protease activity of SltB. Three forms of the SltA transcription factor coexist in cells: a full-length, 78-kDa version and a processed, 32-kDa form, which is found in phosphorylated and unphosphorylated states. The SltA32kDa version mediates transcriptional regulation of sltB and, putatively, genes required for tolerance to cation stress and alkalinity. The full-length form, SltA78kDa, apparently has no transcriptional function. In the absence of SltB, only the primary product of SltA is detectable, and its level equals that of SltA78kDa. Mutations in sltB selected as suppressors of null vps alleles and resulting in cation/alkalinity sensitivity either reduced or eliminated SltA proteolysis. There is no evidence for cation or alkalinity regulation of SltB cleavage, but activation of sltB expression requires SltA. This work identifies the molecular mechanisms governing the Slt pathway.

Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones - a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs - and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolyticactivities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms.

Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolyticactivity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolyticactivity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds

Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases), participate in diverse pathological processes. To simultaneously measure multiple specific protease activities, a series of parallel enzyme reactions combined with a series of inhibitor analyses for proteolyticactivity matrix analysis (PrAMA) are essential but limited due to the sample quantity requirements and the complexity of performing multiple reactions. To address these issues, we developed a pico-injector array to generate 72 different reactions in picoliter-volume droplets by controlling the sequence of combinational injections, which allowed simultaneous recording of a wide range of multiple enzyme reactions and measurement of inhibitor effects using small sample volumes (~10 μL). Multiple MMP activities were simultaneously determined by 9 different substrates and 2 inhibitors using injections from a pico-injector array. Due to the advantages of inhibitor analysis, the MMP/ADAM activities of MDA-MB-231, a breast cancer cell line, were characterized with high MMP-2, MMP-3 and ADAM-10 activity. This platform could be customized for a wide range of applications that also require multiple reactions with inhibitor analysis to enhance the sensitivity by encapsulating different chemical sensors.

The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolyticactivities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell-cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.

Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO{sub 4}/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated {sup 125}I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolyticactivation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

Proteolyticactivity in oat leaf extracts was measured with both azocasein and ribulose bisphosphate carboxylase (Rubisco) as substrates over a wide range of pH (3.0-9.2). With either azocasein or Rubisco activity peaks appeared at pH 4.8, 6.6, and 8.4. An aminopeptidase (AP) which hydrolyzes leucine-nitroanilide was partially purified. Purification consisted of a series of six steps which included ammonium sulfate precipitation, gel filtration, and two ionic exchange chromatographies. The enzyme was purified more than 100-fold. The apparent Km for leucine-nitroanilide is 0.08 millimolar at its pH optimum of 8.4. AP may be a cystein protease since it is inhibited by heavy metals and activated by 2-mercaptoethanol. Isolated chloroplasts were also able to hydrolyze leucine-nitroanilide at a pH optimum of 8.4, indicating that AP could be localized inside the photosynthetic organelles. PMID:16667194

BACKGROUND: Proteoglycans, consisting of glycosaminoglycan (GAG) side chains covalently linked to a protein core, are a major component of the extracellular matrix of the intestinal lamina propria. AIMS: This study investigated the effects of lamina propria T cell activation on the proteoglycan component of the matrix. METHODS: The high degree of sulphation of GAGs means that they are polyanionic and thus can be visualised in tissue sections by means of colloidal-gold labelled cationic probes. RESULTS: In human fetal small intestine there is a dense meshwork of anionic residues in the lamina propria and basement membrane. When explants of human fetal small intestine are cultured ex vivo, and resident lamina propria T cells are activated with pokeweed mitogen, mucosal destruction occurs within three days. This is associated with the rapid loss of anionic sites from the lamina propria. Dermatan sulphate proteoglycan is lost from the tissue and is present at increased concentrations in the organ culture supernatants, indicating that T cell activation has led to solubilisation of lamina propria proteoglycans. Tissue destruction and loss of anionic residues are inhibited in a dose dependent fashion by dexamethasone, and by the protease inhibitor, alpha 2 macroglobulin. CONCLUSIONS: Proteolytic degradation of the lamina propria may therefore be a mechanism by which T cell hypersensitivity injures the intestinal mucosa. Images Figure 1 Figure 4 Figure 5 PMID:8977345

The influence of potential inhibitors naturally present in wine on the proteolyticactivity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine-like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO2 ), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO2 , which acted as a mixed-type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis-Menten equation, was determined to be strongly affected by free SO2 and total phenol level.

The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.

The Bcl-2-associated athanogene (BAG) family is a multifunctional group of proteins involved in numerous cellular functions ranging from apoptosis to tumorigenesis. These proteins are evolutionarily conserved and encode a characteristic region known as the BAG domain. BAGs function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in tumor growth, HIV infection, and neurodegenerative diseases; as a result, the BAGs are attractive targets for therapeutic interventions, and their expression in cells may serve as a predictive tool for disease development. The Arabidopsis genome contains seven homologs of BAG family proteins (Figure 1), including four with a domain organization similar to animal BAGs (BAG1-4). The remaining three members (BAG5-7) contain a predicted calmodulin-binding motif near the BAG domain, a feature unique to plant BAG proteins that possibly reflects divergent mechanisms associated with plant-specific functions. As reported for animal BAGs, plant BAGs also regulate several stress and developmental processes (Figure 2). The recent article by Li et al. focuses on the role of BAG6 in plant innate immunity. This study shows that BAG6 plays a key role in basal plant defense against fungal pathogens. Importantly, this work further shows that BAG6 is proteolyticallyactivated to induce autophagic cell death and resistance in plants. This finding underscores the importance of proteases in the execution of plant cell death, yet little is known about proteases and their substrates in plants. PMID:28358147

Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCδ plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCδ was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCδ fragments to mitochondria in a time-dependent fashion, and the PKCδ inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCδ catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCδ (PKCδ-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCδ-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCδ proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCδ-CRM) or suppression of PKCδ expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolyticallyactivated PKCδ has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells. PMID

ABSTRACT Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. PMID:25336460

Influenza viruses do not encode any proteases and must rely on host proteases for the proteolyticactivation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place. PMID:23365447

Background Vascular endothelial growth factor (VEGF) is a key regulator of physiologic and pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. It is known that cysteine proteases from plants, like bromelain and papain are capable to suppress inflammatory activation. Recent studies have demonstrated that they may interfere with angiogenesis related pathways as well. The aim of this study was to investigate the anti-angiogenic effects of papain on human umbilical vein endothelial cells (HUVEC) in vitro. Methods Cell viability after prolonged treatment with papain was investigated by life cell staining and lactate dehydrogenase release assay. Angiogenic activation was assessed by ELISA against phosphorylated proteins AKT, MEK1/2, ERK1/2, SAPK/JNK and p38-MAPK. Growth inhibition was determined by means of an MTT-assay and cell migration by means of a scratch assay. Capability to form a capillary network was investigated using a tube formation assay. Results Papain did not induce proteolysis or cell detachment of HUVEC in a concentration range between 0 and 25 μg/mL. Four hours treatment with 10 μg/mL papain resulted in a reduced susceptibility of endothelial cells to activation by VEGF as determined by phosphorylation levels of Akt, MEK1/2, SAPK/JNK. Papain exerted a distinct inhibitory effect on cell growth, cell migration and tube formation with inhibition of tube formation detectable at concentrations as low as 1 μg/mL. Bromelain and ficin displayed similar effects with regard to cell growth and tube formation. Conclusion Papain showed a strong anti-angiogenic effect in VEGF activated HUVEC. This effect may be due to interference with AKT, MEK1/2 and SAPK/JNK phosphorylation. Two other plant derived cysteine proteases displayed similar inhibition of HUVEC cell growth and tube formation. These findings indicate that plant proteolytic enzymes may have potential as preventive and therapeutic agents against angiogenesis related human diseases

Influenza viruses do not encode any proteases and must rely on host proteases for the proteolyticactivation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

One of the key mechanisms involved in renal Na+ retention in chronic heart failure (CHF) is activation of epithelial Na+ channels (ENaC) in collecting tubules. Proteolytic cleavage has an important role in activating ENaC. We hypothesized that enhanced levels of proteases in renal tubular fluid activate ENaC resulting in renal Na+ retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF rats compared to sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06 and plasmin 3.57 vs. sham). Similar increases were observed in urinary samples from patients with CHF. Whole-cell patch-clamp was conducted in cultured renal collecting duct M-1 cells to record Na+ currents. Protease-rich urine (from rats and patients with CHF) significantly increased the Na+ inward current in M-1 cells. Two weeks of protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Increased podocyte lesions were observed in the kidneys of rats with CHF by transmission electron microscopy. Consistent with these results, podocyte damage markers desmin and podocin expressions were also increased in rats with CHF (increased ~2 folds). These findings suggest that podocyte damage may lead to increased proteases in the tubular fluid which in turn contributes to the enhanced renal ENaC activity, providing a novel mechanistic insight for Na+ retention commonly observed in CHF. PMID:26628676

Seeds of bread wheat were incubated at 40 degrees C and 100% relative humidity for 0, 3, 4, 6, and 10 days. The effects of accelerated aging on seed germinability and some biochemical properties of flour (carotenoid, free radical, and protein contents and proteolyticactivity) and gluten (free radical content and flexibility) were investigated. Seed germinability decreased during aging, resulting in seed death after 10 days. A progressive decrease of carotenoid content, in particular, lutein, was observed, prolonging the incubation, whereas the free radical content increased in both flour and gluten. A degradation of soluble and storage proteins was found, associated with a marked increase of proteolyticactivity and a loss of viscoelastic properties of gluten. On the contrary, puroindolines were quite resistant to the treatment. The results are discussed in comparison with those previously obtained during accelerated aging of durum wheat seeds.

We have observed two proteolyticactivities in cell lysates from the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation, and other peptides containing similar sequences. Both activities are inhibited by Pefabloc, a serine protease inhibitor, while one of the activities is inhibited by either EDTA or aprotinin. The protease inhibitors pepstatin, bestatin, E-64, leupeptin and phosphoramidon do not block either of these proteolyticactivities.

Surface structures on guinea pig ventricular myocytes were protected from proteolytic enzyme activity with 100 KIU.ml-2 aprotinin during mechanical disaggregation. Intact myocytes, approximately 7.5 X 10(6) cells.g-1 ventricular wet weight, were separated from debris and damaged cells using Cytodex I tissue culture supports. Cellular ultrastructure did not differ from that observed in intact tissue. Neither spontaneous contractions nor contracture were ever observed in these myocytes in calcium concentrations of 10 mmol.litre-1. Dinitrophenol (0.2 mmol. litre-1) uncoupled respiration in the myocytes but only after the sarcolemma had been disrupted with Triton X100. The adenosine diphosphate to oxygen ratio of mitochondria isolated from the myocytes was 2.4(0.2) and the respiratory control index 2.6(0.3). Calcium (1.8 mmol.litre-1) increased oxygen uptake in the presence of 10 mmol.litre-1 pyruvate or 11 mmol.litre-1 glucose but not 17 mmol. litre-1 succinate. Succinate dependent oxygen consumption was greater than pyruvate dependent oxygen consumption (1090.0(190.0) and 40.1(0.8) nl.min-1.mg-1 protein respectively). The Crabtree effect was present. Oxidative metabolism was normal in cells stored at 10 degrees C for seven days but deteriorated rapidly thereafter. The results indicate that myocytes disaggregated by this procedure retain many of the morphological and metabolic characteristics of intact cardiac muscle cells and are relatively homogeneous with respect to calcium tolerance and metabolic function.

Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolyticactivity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially. PMID:23949661

This laboratory exercise uses a problem-based approach to expose students to some basic concepts relating to proteins and enzymes. One of the main applications of enzymes at the industrial level is their use in the detergent market. The students examine a detergent sample to ascertain whether proteolytic enzymes are a component and, if so, which…

Alterations of the intracellular ubiquitin-proteasome pathway are found in neurodegenerative and inflammatory disorders of the central nervous system, as well as in its malignancies. Inhibitory substrates of the proteasomes represent promising approaches to control autoimmune inflammations and induction of apoptosis in cancer cells. Extracellular circulating proteasomes are positively correlated to outcome prognosis in hematogenic neoplasias and the outcome in critically ill patients. Previously, we reported raised levels of proteolyticactive 20S proteasomes in the extracellular alveolar space in patients with acute respiratory distress syndrome (ARDS). For the cerebrospinal fluid, we assumed that extracellular circulating proteasomes with enzymatic activity can be found, too. Cerebrospinal fluid (CSF) samples of twenty-six patients (14 females, 12 males), who underwent diagnostic spinal myelography, were analyzed for leukocyte cell count, total protein content, lactate and interleukine-6 (Il-6) concentrations. CSF samples were analyzed for concentration and enzymatic activity of extracellular 20S proteasomes (fluorescenic substrate cleavage; femtokatal). Blood samples were analyzed with respect to concentration of extracellular circulating proteasomes. Choroidal plexus was harvested at autopsies and examined with immunoelectron microscopy (EM) for identification of possible transportation mechanisms. Statistical analysis was performed using SPSS (18.0.3). In all patients, extracellular proteasome was found in the CSF. The mean concentration was 24.6 ng/ml. Enzymatic activity of the 20S subunits of proteasomes was positively identified by the fluorescenic subtrate cleavage at a mean of 8.5 fkat/ml. Concentrations of extracellular proteasomes in the CSF, total protein content and Il-6 were uncorrelated. Immunoelectron microscopy revealed merging vesicles of proteasomes with the outer cell membrane suggestive of an exozytic transport mechanism. For the first time

The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity. PMID:23656734

We improved the enzymatic properties of the oxidatively stable alkaline serine protease KP-43 through protein engineering to make it more suitable for use in laundry detergents. To enhance proteolyticactivity, the gene encoding KP-43 was mutagenized by error-prone PCR. Screening identified a Tyr195Cys mutant enzyme that exhibited increased specific activity toward casein between pH 7 and 11. At pH 10, the mutant displayed 1.3-fold higher specific activity for casein compared to the wild-type enzyme, but the activity of the mutant was essentially unchanged toward several synthetic peptides. Furthermore, the Tyr195Cys mutation significantly increased thermal stability and surfactant stability of the enzyme under oxidizing conditions. Examination of the crystal structure of KP-43 revealed that Tyr195 is a solvent exposed residue that forms part of a flexible loop that binds a Ca(2+) ion. This residue lies 15-20Å away from the residues comprising the catalytic triad of the enzyme. These results suggest that the substitution at position 195 does not alter the structure of the active center, but instead may affect a substrate-enzyme interaction. We propose that the Tyr195Cys mutation enhances the interaction with Ca(2+) and affects the packing of the Ca(2+) binding loop, consequently increasing protein stability. The simultaneously increased proteolyticactivity, thermal stability, and surfactant stability of the Tyr195Cys mutant enzyme make the protein an ideal candidate for laundry detergent application.

Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolyticactivity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolyticactivities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs. PMID:27022410

Arthrobotrys musiformis is a nematophagous fungus with potential for the biological control of Haemonchus contortus larvae. This study aimed to identify and demonstrate the proteolyticactivity of extracellular products from A musiformis cultured in a liquid medium against H contortus infective larvae. A musiformis was cultured on a solid medium and further grown in a liquid medium, which was then processed through ion exchange and hydrophobic interaction chromatography. The proteolyticactivity of the purified fraction was assayed with either gelatin or bovine serum albumin as substrate. Optimum proteolyticactivity was observed at pH 8 and a temperature of 37°C. Results obtained with specific inhibitors suggest the enzyme belongs to the serine-dependent protease family. The purified fraction concentrate from A musiformis was tested against H contortus infective larvae. A time-dependent effect was observed with 77 per cent immobility after 48 hours incubation, with alteration of the sheath. It is concluded that A musiformis is a potential candidate for biological control because of its resistant structures and also because of its excretion of extracellular products such as proteases. The present study contributes to the identification of one of the in vitro mechanisms of action of Amusiformis, namely the extracellular production of proteases against H contortus infective larvae. More investigations should be undertaken into how these products could be used to decrease the nematode population in sheep flocks under field conditions, thereby improving animal health while simultaneously diminishing the human and environmental impact of chemical-based drugs. PMID:26392902

Following collection of seawater samples during an Arctic Chukchi Sea expedition cruise of the Korean icebreaker Araon in 2012, a total of 15,696 bacteria were randomly isolated from Marine Broth 2216 agar plates. Of these, 2,526 (16%) showed proteolyticactivity and were identified as mainly Alteromonas (31%), Staphylococcus (27%), and Pseudoalteromonas (14%). Among the proteolytic strains, seven were selected based on their significant ability to grow and produce a halo on skim milk plates at low temperatures (<5°C) owing to cold-active proteases. These strains were affiliated with the genus Pseudoalteromonas and were divided into three groups based on phylogenetic analysis of the 16S rRNA genes. Profiling cell membrane fatty acids confirmed the 16S rRNA-based differentiation and revealed the accordance between the two analyses. Seven genes for serine protease precursors were amplified from the corresponding strains, and based on sequence similarities, these genes were divided into three groups that were identical to those identified by the 16S rRNA phylogenetic analysis. Three protease genes from the representative strains of each group were composed of 2,127-2,130 bp, encoding 708-709 amino acids, and these genes yielded products with calculated molecular weights of approximately 72.3-72.8 kDa. Amino acid sequence analysis suggested that the precursors are members of the subtilase serine endo- and exo-peptidase clan and contain four domains (signal peptide, N-terminal prosequence, catalytic domain, and two pre-peptidase C-terminal domains). Upon expression in E. coli, each recombinant protease exhibited proteolyticactivity on zymogram gels.

Expression of hemolytic and proteolyticactivities throughout the growth cycle was investigated with two enterotoxic aeromonad strains assigned to the species Aeromonas hydrophila and Aeromonas veronii biovar sobria. Although growth kinetic data were dependent on strain, temperature, and substrate, maximum populations attained were higher than 9 log CFU/ml in aerated tryptone soya broth plus yeast extract (TSBYE) and salmon extract within the range 4 to 28 degrees C. For both strains in TSBYE, variable amounts of hemolytic activity were first detected at any temperature when aeromonad counts were over 9 log CFU/ml. Afterwards, this activity increased up to similar levels (109 to 112 hemolytic units per ml) without a significant increase in populations. Salmon extract supported hemolysin synthesis at 28 but not 4 degrees C. Proteolyticactivity of the A. hydrophila strain was only expressed in salmon extract at 28 degrees C, whereas A. veronii biovar sobria did at 28 degrees C in both substrates and at 10 degrees C in TSBYE.

Atrial natriuretic peptide (ANP) is rapidly cleared and degraded in vivo. Nonguanylate-cyclase receptors (C-ANPR) and a metalloproteinase, neutral endopeptidase (EC 3.4.24.11) (NEP 24.11), are thought to be responsible for its metabolism. We investigated the mechanisms of ANP degradation by an endothelial-derived cell line, CPA47. CPA47 cells degraded 88 percent of 125I-ANP after 1 h at 37 degrees C as determined by HPLC. Medium preconditioned by these cells degraded 41 percent of the 125I-ANP, and this activity was inhibited by a divalent cation chelator, EDTA. Furthermore, a cell-surface proteolyticactivity degraded 125I-ANP in the presence of EDTA when receptor-mediated endocytosis was inhibited either by low temperature (4 degrees C) or by hyperosmolarity at 37 degrees C. The metalloproteinase, NEP 24.11, is unlikely to be the cell-surface peptidase because 125I-ANP is degraded by CPA47 cells at 4 degrees C in the presence of 5 mM EDTA. These data indicate that CPA47 cells can degrade ANP by a novel divalent cation-independent cell-surface proteolyticactivity.

Membrane active peptides are of large interest for development of drug delivery vehicles and therapeutics for treatment of multiple drug resistant infections. Lack of specificity can be detrimental and finding routes to tune specificity and activity of membrane active peptides is vital for improving their therapeutic efficacy and minimize harmful side effects. We describe a de novo designed membrane active peptide that partition into lipid membranes only when specifically and covalently anchored to the membrane, resulting in pore-formation. Dimerization with a complementary peptide efficiently inhibits formation of pores. The effect can be regulated by proteolytic digestion of the inhibitory peptide by the matrix metalloproteinase MMP-7, an enzyme upregulated in many malignant tumors. This system thus provides a precise and specific route for tuning the permeability of lipid membranes and a novel strategy for development of recognition based membrane active peptides and indirect enzymatically controlled release of liposomal cargo.

Accumulation of the amyloid-beta (Abeta) peptide depends on both its generation and clearance. To better define clearance pathways, we have evaluated the role of the tissue plasminogen activator (tPA)-plasmin system in Abeta degradation in vivo. In two different mouse models of Alzheimer's disease, chronically elevated Abeta peptide in the brain correlates with the upregulation of plasminogen activator inhibitor-1 (PAI-1) and inhibition of the tPA-plasmin system. In addition, Abeta injected into the hippocampus of mice lacking either tPA or plasminogen persists, inducing PAI-1 expression and causing activation of microglial cells and neuronal damage. Conversely, Abeta injected into wild-type mice is rapidly cleared and does not cause neuronal degeneration. Thus, the tPA-plasmin proteolytic cascade aids in the clearance of Abeta, and reduced activity of this system may contribute to the progression of Alzheimer's disease.

A transposition mutant of Staphylococcus aureus was selected from the parent strain MT23142, a derivative of strain 8325. The site of transposition was near the 5′ terminus of the gene arlS. ArlS exhibits strong similarities with histidine protein kinases. Sequence analysis suggested that arlS forms an operon with upstream gene arlR. The predicted product of arlR is a member of the OmpR-PhoB family of response regulators. The arlS mutant formed a biofilm on a polystyrene surface unlike the parent strain and the complemented mutant. Biofilm formation was associated with increased primary adherence to polystyrene, whereas cellular adhesion was only slightly decreased. In addition, the arlS mutant exhibited increased autolysis and altered peptidoglycan hydrolase activity compared to the parental strain and to the complemented mutant. As it has been shown for coagulase-negative staphylococci that some autolysins are able to bind polymer surfaces, these data suggest that the two-component regulatory system ArlS-ArlR may control attachment to polymer surfaces by affecting secreted peptidoglycan hydrolase activity. Finally, the arlS mutant showed a dramatic decrease of extracellular proteolyticactivity, including serine protease activity, in comparison to the wild-type strain and the complemented mutant, and cells grown in the presence of phenylmethylsulfonyl fluoride (a serine protease inhibitor) showed an increased autolysin activity. Since the locus arlR-arlS strikingly modifies extracellular proteolyticactivity, this locus might also be involved in the virulence of S. aureus. PMID:10869073

Redox potential and proteolysis determine protein networks in doughs and thus dough rheology as well as the structure of baked goods. Namely, gluten-free bakery products needs structural improvements but little is known about these parameters in gluten free dough systems. In this work the influence of lactic acid bacteria (LAB) on redox status and proteolysis of buckwheat sourdoughs was investigated. An increase of free thiol groups was detected as redox potential was decreasing during fermentation. Thiol content at 8 h was higher in doughs fermented with strains with high reductive activity, such as Weissella (W.) cibaria in comparison to Pediococcus (P.) pentosaceus, which exhibited a lower reducing activity. At 24 h each fermentation showed a similar content of free thiol groups. Endogenous buckwheat proteases were characterized using various protease inhibitors in buckwheat doughs. Until pH3.1 a proteolysis increase was monitored in doughs. Employed LAB didn't show any detectable extracellular proteolyticactivity. Flour proteases are thus responsible for protein breakdown, and this was demonstrated comparing free amino nitrogen (FAN) values and protein electrophoretic patterns of sourdough fermentations with chemical acidified (CA) doughs. FAN content at 24 h using P. pentosaceus, proteolytic comparative strain of Enterococcus faecalis, W. cibaria, mixed culture (containing P. pentosaceus and W. cibaria), CA and CA doughs containing glutathione (GSH) reached 45.9±1.3, 42.4±1.3, 40±1, 31±2, 29±2 and 17.8±3.9 mmol kg(-1) flour, respectively. Proteolysis was mainly influenced by pH and incubation time. The addition of GSH showed a decrease of proteolysis and of free amino acids. CA doughs showed a higher total free amino acids content than sourdough fermented with LAB indicating their metabolization. Fermentations with high FAN values exhibited lower band intensity (analyzed under reducing condition) in electrophoretic patterns. These results show that

Lactic acid bacteria (LAB) can be isolated from different sources such as milk and cheese, and the lipolytic, proteolytic and glycolytic enzymes of LAB are important in cheese preservation and in flavour production. Moreover, LAB produce several antimicrobial compounds which make these bacteria interesting for food biopreservation. These characteristics stimulate the search of new strains with technological potential. From 156 milk and cheese samples from cow, buffalo and goat, 815 isolates were obtained on selective agars for LAB. Pure cultures were evaluated for antimicrobial activities by agar antagonism tests and for proteolyticactivity on milk proteins by cultivation on agar plates. The most proteolytic isolates were also tested by cultivation in skim milk followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fermented milk. Among the 815 tested isolates, three of them identified as Streptococcus uberis (strains FT86, FT126 and FT190) were bacteriocin producers, whereas four other ones identified as Weissella confusa FT424, W. hellenica FT476, Leuconostoc citreum FT671 and Lactobacillus plantarum FT723 showed high antifungal activity in preliminary assays. Complementary analyses showed that the most antifungal strain was L. plantarum FT723 that inhibited Penicillium expansum in modified MRS agar (De Man, Rogosa, Sharpe, without acetate) and fermented milk model, however no inhibition was observed against Yarrowia lipolytica. The proteolytic capacities of three highly proteolytic isolates identified as Enterococcus faecalis (strains FT132 and FT522) and Lactobacillus paracasei FT700 were confirmed by SDS-PAGE, as visualized by the digestion of caseins and whey proteins (β-lactoglobulin and α-lactalbumin). These results suggest potential applications of these isolates or their activities (proteolyticactivity or production of antimicrobials) in dairy foods production.

ADAMTS13 is a plasma metalloproteinase that cleaves large multimeric forms of von Willebrand factor (VWF) to smaller, less adhesive forms. ADAMTS13 activity is reduced in systemic inflammatory syndromes, but the cause is unknown. Here, we examined whether neutrophil-derived oxidants can regulate ADAMTS13 activity. We exposed ADAMTS13 to hypochlorous acid (HOCl), produced by a myeloperoxidase-H2O2-Cl(-) system, and determined its residual proteolyticactivity using both a VWF A2 peptide substrate and multimeric plasma VWF. Treatment with 25 nm myeloperoxidase plus 50 μm H2O2 reduced ADAMTS13 activity by >85%. Using mass spectrometry, we demonstrated that Met(249), Met(331), and Met(496) in important functional domains of ADAMTS13 were oxidized to methionine sulfoxide in an HOCl concentration-dependent manner. The loss of enzyme activity correlated with the extent of oxidation of these residues. These Met residues were also oxidized in ADAMTS13 exposed to activated human neutrophils, accompanied by reduced enzyme activity. ADAMTS13 treated with either neutrophil elastase or plasmin was inhibited to a lesser extent, especially in the presence of plasma. These observations suggest that oxidation could be an important mechanism for ADAMTS13 inactivation during inflammation and contribute to the prothrombotic tendency associated with inflammation.

Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo. It was thought that intracellular processing was restricted to the TGN (trans-Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)-Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late embryonic and postnatal rat tail and postnatal mouse tail tendon, C-proteinase activity was detected in TGN and pre-TGN compartments. Preventing activation of the procollagen N- and C-proteinases with the furin inhibitor Dec-RVKR-CMK (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) indicated that only a fraction of intracellular procollagen cleavage was mediated by newly activated proteinases. In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell-matrix interface.

Background The mechanisms of progressive dopaminergic neuronal loss in Parkinson’s disease (PD) remain poorly understood, largely due to the complex etiology and multifactorial nature of disease pathogenesis. Several lines of evidence from human studies and experimental models over the last decade have identified neuroinflammation as a potential pathophysiological mechanism contributing to disease progression. Tumor necrosis factor α (TNF) has recently emerged as the primary neuroinflammatory mediator that can elicit dopaminergic cell death in PD. However, the signaling pathways by which TNF mediates dopaminergic cell death have not been completely elucidated. Methods In this study we used a dopaminergic neuronal cell model and recombinant TNF to characterize intracellular signaling pathways activated during TNF-induced dopaminergic neurotoxicity. Etanercept and neutralizing antibodies to tumor necrosis factor receptor 1 (TNFR1) were used to block TNF signaling. We confirmed the results from our mechanistic studies in primary embryonic mesencephalic cultures and in vivo using the stereotaxic lipopolysaccharide (LPS) model of nigral dopaminergic degeneration. Results TNF signaling in dopaminergic neuronal cells triggered the activation of protein kinase Cδ (PKCδ), an isoform of the novel PKC family, by caspase-3 and caspase-8 dependent proteolytic cleavage. Both TNFR1 neutralizing antibodies and the soluble TNF receptor Etanercept blocked TNF-induced PKCδ proteolyticactivation. Proteolyticactivation of PKCδ was accompanied by translocation of the kinase to the nucleus. Notably, inhibition of PKCδ signaling by small interfering (si)RNA or overexpression of a PKCδ cleavage-resistant mutant protected against TNF-induced dopaminergic neuronal cell death. Further, primary dopaminergic neurons obtained from PKCδ knockout (−/−) mice were resistant to TNF toxicity. The proteolyticactivation of PKCδ in the mouse substantia nigra in the neuroinflammatory LPS

Temporal responses of indigenous bacterial populations and proteolytic enzyme (i.e., aminopeptidase) activities in the bacterioplankton assemblages from 3 separate freshwater environments were examined after exposure to various zinc (Zn) concentrations under controlled microcosm conditions. Zn concentrations (ranging from 0 to 10 μmol/L) were added to water samples collected from the Kalamazoo River, Rice Creek, and Huron River and examined for bacterial abundance and aminopeptidase activities at various time intervals over a 48 h incubation period in the dark. The results showed that the Zn concentrations did not significantly influence total bacterial counts directly; however, aminopeptidase activities varied significantly to increasing zinc treatments over time. Also, analysis of variance and linear regression analyses revealed significant positive relationships between bacterial numbers and their hydrolytic enzyme activities, suggesting that both probably co-vary with increasing Zn concentrations in aquatic systems. The results from this study serve as additional evidence of the ecological role of Zn as an extracellular peptidase cofactor on the dynamics of bacterial assemblages in aquatic environments.

The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.''

Actinidin, a member of the papain-like family of cysteine proteases, is abundant in kiwifruit. To date, a few studies have been provided to investigate the proteolyticactivity and substrate specificity of actinidin on native proteins. Herein, the proteolyticactivity of actinidin was compared to papain on several different fibrous and globular proteins under neutral, acidic and basic conditions. The digested samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry to assess the proteolytic effect. Furthermore, the levels of free amino nitrogen (FAN) of the treated samples were determined using the ninhydrin colorimetric method. The findings showed that actinidin has no or limited proteolytic effect on globular proteins such as immunoglobulins including sheep IgG, rabbit IgG, chicken IgY and fish IgM, bovine serum albumin (BSA), lipid transfer protein (LTP), and whey proteins (α-lactalbumin and β-lactoglobulin) compared to papain. In contrast to globular proteins, actinidin could hydrolyze collagen and fibrinogen perfectly at neutral and mild basic pHs. Moreover, this enzyme could digest pure α-casein and major subunits of micellar casein especially in acidic pHs. Taken together, the data indicated that actinidin has narrow substrate specificity with the highest enzymatic activity for the collagen and fibrinogen substrates. The results describe the actinidin as a mild plant protease useful for many special applications such as cell isolation from different tissues and some food industries as a mixture formula with other relevant proteases.

Tissue from pons medulla of rats exposed in vivo to various levels of ozone was assayed for calpain and cathepsin D activity. Chronic exposure to ozone increased calpain activity, which was 35% to 46% higher in the homogenates of animals exposed to 1.0 ppm ozone than in those of animals exposed to 0.5 ppm ozone or of controls. An increase in activity of 26% was also observed in the soluble supernatant. The increase in activity did not seem to be caused by ozone effects on calpastatin. Addition of 32 mM carnitine to the incubation mixture increased total activity 3-4 fold, making the differences in activity proportionately smaller. Cathepsin D activity was little altered. Changes in calpain activity and in the generation of free oxygen radicals have been implicated in the aging process, long-term exposure to ozone may magnify changes. Ozone exposure may cause changes in brain protein metabolism. 15 refs., 2 tabs.

Over the last four centuries, cocoa and chocolate have been described as having potential medicinal value. As of today, Theobroma cacao L. (Sterculiaceae) and its products are consumed worldwide. They are of great research interest because of the concentration dependent antioxidant as well as pro-oxidant properties of some of their polyphenolic constituents, specially procyanidins and flavan-3-ols such as catechin. This study was aimed at investigating the cellular and molecular changes associated with cytotoxicity, caused due pro-oxidant activity of cocoa catechins and procyanidins, in ovarian cancer cell lines. Extract of non-alkalized cocoa powder enriched with catechins and procyanidins was used to treat human epithelial ovarian cancer cell lines OAW42 and OVCAR3 at various concentrations ≤1000μg/mL. The effect of treatment on intracellular reactive oxygen species (ROS) levels was determined. Apoptotic cell death, post treatment, was evaluated microscopically and using flow cytometry by means of annexin-propidium iodide (PI) dual staining. Levels of active caspase-3 as a pro-apoptotic marker and matrix metalloproteinase 2 (MMP2) as an invasive potential marker were detected using Western blotting and gelatin zymography. Treatment with extract caused an increase in intracellular ROS levels in OAW42 and OVCAR3 cell lines. Bright field and fluorescence microscopy of treated cells revealed apoptotic morphology and DNA damage. Increase in annexin positive cell population and dose dependent upregulation of caspase-3 confirmed apoptotic cell death. pro-MMP2 was found to be downregulated in a dose dependent manner in cells treated with the extract. Treated cells also showed a reduction in MMP2 activity. Our data suggests that cocoa catechins and procyanidins are cytotoxic to epithelial ovarian cancer, inducing apoptotic morphological changes, DNA damage and caspase-3 mediated cell death. Downregulation of pro-MMP2 and reduction in active MMP2 levels imply a decrease

Collagen-binding activity (CBA) and FRETS-VWF73 assays are widely adopted methods for the measurement of the plasmatic activity of ADAMTS13, the von Willebrand factor (VWF) cleaving-protease. Accurately assessing the severe deficiency of ADAMTS13 is important in the management of thrombotic thrombocytopenic purpura (TTP). However, non-concordant results between the two assays have been reported in a small but relevant percentage of TTP cases. We investigated whether CBA or FRETS-VWF73 assay reflects ADAMTS13 proteolyticactivity in acquired TTP patients with non-concordant measurements. Twenty plasma samples with non-concordant ADAMTS13 activity results, <10% using FRETS-VWF73 and ≥20% using CBA, and 11 samples with concordant results, <10% using either FRETS-VWF73 and CBA assays, were analysed. FRETS-VWF73 was performed in the presence of 1.5 M urea. ADAMTS13 activities were also measured under flow conditions and the VWF multimer pattern was defined in order to verify the presence of ultra-large VWF due to ADAMTS13 deficiency. In FRETS-VWF73 assay with 1.5 M urea, ADAMTS13 activity significantly increased in roughly 50% of the samples with non-concordant results, whereas it remained undetectable in all samples with concordant measurements. Under flow conditions, all tested samples showed reduced ADAMTS13 activity. Finally, samples with non-concordant results showed a ratio of high molecular weight VWF multimers higher than normal. Our results support the use of FRETS-VWF73 over CBA assay for the assessment of ADAMTS13 severe deficiency and indicate urea as one cause of the observed differences.

Lon proteases degrade defective or denature proteins as well as some folded proteins for the control of cellular protein quality. There are two types of Lon proteases, LonA and LonB. Each consists of two functional components: a protease component and an ATPase associated with various cellular activities (AAA+ module). Here, we report the 2.03 -resolution crystal structure of the isolated AAA+ module (iAAA+ module) of LonB from Thermococcus onnurineus NA1 (TonLonB). The iAAA+ module, having no bound nucleotide, adopts a conformation virtually identical to the ADP-bound conformation of AAA+ modules in the hexameric structure of TonLonB; this provides insights into the ATP-independent proteolyticactivity observed in a LonB protease. Structural comparison of AAA+ modules between LonA and LonB revealed that the AAA+ modules of Lon proteases are separated into two distinct clades depending on their structural features. The AAA+ module of LonB belongs to the -H2 & Ins1 insert clade (HINS clade)- defined for the first time in this study, while the AAA+ module of LonA is a member of the HCLR clade.

Strains of Saccharomyces cerevisiae isolated from Italian salami were screened for proteolyticactivity in a model system containing sarcoplasmic (SMS) or myofibrillar (MMS) proteins, at 20°C for 14days, to evaluate the possible influence on the proteolysis of fermented sausages. SDS-PAGE revealed that 14 of the most osmotolerant strains were responsible for the extensive hydrolysis of the main myofibrillar proteins, while only one strain was able to hydrolyze sarcoplasmic proteins. Free amino acids (FAA) accumulated during proteolysis were strain-dependent with different patterns from sarcoplasmic or myofibrillar protein fraction. In general, proteolysis lead Cys, Glu, Lys and Val as the most abundant FAA in the inoculated MMS samples. Volatile compound analysis, determined by SPME-GC-MS, evidenced 3-methyl butanol in MMS, and 2-methyl propanol and 3-methyl-1-butanol in SMS as major compounds. Our findings highlight that S. cerevisiae could influence the composition in amino acids and volatile compounds in fermented sausages, with a strain-dependent activity.

Previous theoretical studies have determined the intermolecular interactions between Mucor pusillus pepsin (MPP) and the key domain of κ-casein, with the aim to understand the mechanism of milk clotting in the specific hydrolysis of κ-casein by MPP for cheese making. Here, we combined the docking model with site-directed mutagenesis to further investigate the functional roles of amino acid residues in the active site of MPP. T218S replacement caused a low thermostability and moderate increase in the clotting activity. Mutations of three amino acid residues, T218A and T218S in S2 region and L287G in S4 region, led to a significant decrease in proteolyticactivity. For T218S and L287G, an increase in the ratio of clotting activity to proteolyticactivity (C/P) was observed, in particular 3.34-fold increase was found for T218S mutants. Structural analysis of the binding mode of MPP and chymosin splitting domain (CSD) of κ-casein indicated that T218S plays a critical role in forming a hydrogen bond with the hydroxyl group of Ser(104) around the MPP-sensitive Phe(105)-Met(106) peptide bond of κ- casein and L287G is partially responsible for CSD accommodation in a suitable hydrophobic environment. These data suggested that T218S mutant could serve as a promising milk coagulant that contributes to an optimal flavor development in mature cheese.

Accumulation of amyloid-β peptide (Aβ), the neurotoxic peptide implicated in the pathogenesis of Alzheimer's disease (AD), has been shown in brain mitochondria of AD patients and of AD transgenic mouse models. The presence of Aβ in mitochondria leads to free radical generation and neuronal stress. Recently, we identified the presequence protease, PreP, localized in the mitochondrial matrix in mammalian mitochondria as the novel mitochondrial Aβ-degrading enzyme. In the present study, we examined PreP activity in the mitochondrial matrix of the human brain's temporal lobe, an area of the brain highly susceptible to Aβ accumulation and reactive oxygen species (ROS) production. We found significantly lower hPreP activity in AD brains compared with non-AD age-matched controls. By contrast, in the cerebellum, a brain region typically spared from Aβ accumulation, there was no significant difference in hPreP activity when comparing AD samples to non-AD controls. We also found significantly reduced PreP activity in the mitochondrial matrix of AD transgenic mouse brains (Tg mAβPP and Tg mAβPP/ABAD) when compared to non-transgenic aged-matched mice. Furthermore, mitochondrial fractions isolated from AD brains and Tg mAβPP mice had higher levels of 4-hydroxynonenal, an oxidative product, as compared with those from non-AD and nonTg mice. Accordingly, activity of cytochrome c oxidase was significantly reduced in the AD mitochondria. These findings suggest that decreased PreP proteolyticactivity, possibly due to enhanced ROS production, contributes to Aβ accumulation in mitochondria leading to the mitochondrial toxicity and neuronal death that is exacerbated in AD. Clearance of mitochondrial Aβ by PreP may thus be of importance in the pathology of AD.

Enzymatic activity of Ca2+-dependent calpain proteases as well as the content and gene expression of μ-calpain (activated by micromolar calcium ion concentrations), calpastatin (inhibitor of calpains), and titin (substrate for calpains) were investigated in cardiac muscles of rats subjected to chronic alcoholization for 3 and 6 months. There was no increase in the "heart weight/body weight" parameter indicating development of heart hypertrophy in the alcoholized rats, while a decreasing trend was observed for this parameter in the rats after 6-month modeling of alcoholic cardiomyopathy, which indicated development of atrophic changes in the myocardium. Fluorometric measurements conducted using the Calpain Activity Assay Kit did not reveal any changes in total calpain activity in protein extracts of cardiac muscles of the rats alcoholized for 3 and 6 months. Western blot analysis did not show reliable changes in the contents of μ-calpain and calpastatin, and SDS-PAGE did not reveal any decrease in the titin content in the myocardium of rats after the chronic alcohol intoxication. Autolysis of μ-calpain was also not verified, which could indicate that proteolyticactivity of this enzyme in myocardium of chronically alcoholized rats is not enhanced. Using Pro-Q Diamond staining, changes in phosphorylation level of titin were not detected in cardiac muscle of rats after chronic alcoholization during three and six months. A decrease in µ-calpain and calpastatin mRNA content (~1.3-fold, p ≤ 0.01 and ~1.9-fold, p ≤ 0.01, respectively) in the myocardium of rats alcoholized for 3 months and decrease in calpastatin mRNA (~1.4-fold, p ≤ 0.01) in animals alcoholized for 6 months was demonstrated using real-time PCR. These results indicate negative effect of chronic alcohol intoxication on expression of the abovementioned genes.

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

Herein, we have studied a consanguineous Egyptian family with two children diagnosed with severe autosomal recessive osteogenesis imperfecta (AR-OI) and a large umbilical hernia. Homozygosity mapping in this family showed lack of linkage to any of the previously known AR-OI genes, but revealed a 10.27 MB homozygous region on chromosome 8p in the two affected sibs, which comprised the procollagen I C-terminal propeptide (PICP) endopeptidase gene BMP1. Mutation analysis identified both patients with a Phe249Leu homozygous missense change within the BMP1 protease domain involving a residue, which is conserved in all members of the astacin group of metalloproteases. Type I procollagen analysis in supernatants from cultured fibroblasts demonstrated abnormal PICP processing in patient-derived cells consistent with the mutation causing decreased BMP1 function. This was further confirmed by overexpressing wild type and mutant BMP1 longer isoform (mammalian Tolloid protein [mTLD]) in NIH3T3 fibroblasts and human primary fibroblasts. While overproduction of normal mTLD resulted in a large proportion of proα1(I) in the culture media being C-terminally processed, proα1(I) cleavage was not enhanced by an excess of the mutant protein, proving that the Phe249Leu mutation leads to a BMP1/mTLD protein with deficient PICP proteolyticactivity. We conclude that BMP1 is an additional gene mutated in AR-OI. PMID:22052668

This paper presents an algorithm which is able to extract discriminant rules from oligopeptides for protease proteolytic cleavage activity prediction. The algorithm is developed using genetic programming. Three important components in the algorithm are a min-max scoring function, the reverse Polish notation (RPN) and the use of minimum description length. The min-max scoring function is developed using amino acid similarity matrices for measuring the similarity between an oligopeptide and a rule, which is a complex algebraic equation of amino acids rather than a simple pattern sequence. The Fisher ratio is then calculated on the scoring values using the class label associated with the oligopeptides. The discriminant ability of each rule can therefore be evaluated. The use of RPN makes the evolutionary operations simpler and therefore reduces the computational cost. To prevent overfitting, the concept of minimum description length is used to penalize over-complicated rules. A fitness function is therefore composed of the Fisher ratio and the use of minimum description length for an efficient evolutionary process. In the application to four protease datasets (Trypsin, Factor Xa, Hepatitis C Virus and HIV protease cleavage site prediction), our algorithm is superior to C5, a conventional method for deriving decision trees.

Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery (i.e., 'bounce-back') of proteasome activity. We previously demonstrated that the transcription factor Nrf1/NFE2L1 mediates this homeostatic response in mammalian cells. We show here that Nrf1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated Nrf1 is degraded promptly by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated Nrf1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER membrane. Importantly, this cleavage event is essential for Nrf1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol. DOI: http://dx.doi.org/10.7554/eLife.01856.001.

We investigated liver protease activity in force-fed and non-force-fed ducks using zymography gels to better understand mechanisms underlying liver steatosis in palmipeds. Male Muscovy and Pekin ducks were slaughtered before and after a short period (13 d) while they were conventionally fed or force fed. The force-fed regimen contained a high level of carbohydrates and was delivered in large doses. Main hepatic proteases (matrix metalloprotease-2, calpains, and cathepsins) were extracted from raw liver and specifically activated within electrophoretic gels. Both force-fed Muscovy and Pekin ducks presented higher liver weights and BW associated with lower matrix metalloprotease-2 and m-calpain hepatic activities. On the other hand, hepatic cathepsin activity was not affected by force feeding. It was concluded that Muscovy and Pekin duck hepatic proteases are affected similarly by the force feeding. Thus, this cannot explain differences observed between Muscovy and Pekin ducks regarding their ability to develop hepatic steatosis generally reported in literature.

Caspases execute programmed cell death, where low levels of caspase activity are linked to cancer. Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process, however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. By utilizing the optical properties of plasmon coupling, peptide-linked gold nanoparticles were developed to enable single molecule imaging of caspase-3 activity in two different cancer systems. Au crown nanoparticles were assembled in a multimeric fashion to overcome the high and heterogeneous background scattering of live cells. In a colon cancer (SW620) cell line challenged with tumor necrosis factor-alpha (TNF-alpha), single molecule trajectories show early stage caspase-3 activation within minutes, which was not detectable by ensemble assays until 23 hours. Variability in caspase-3 activation among the population of cells was identified and likely a result of each cell's specific resistance to death receptor-induced apoptosis. Following these studies, improvements by way of sensitivity and selectivity were tailored into an improved nanosensor construct. Au nanoshell dimers were prepared as a comparably bright construct with 1) reduced heterogeneity compared to the synthesis of the crown nanoparticles and 2) a peptide sequence highly selective for caspase-3. Chronic myeloid leukemia (CML) K562 cells were assessed for their early apoptotic response upon treatment with dasatinib, a clinically approved tyrosine kinase inhibitor that specifically targets BCR-ABL. It has been demonstrated that inhibition of BCR-ABL by dasatinib commits K562 cells to apoptosis. Single molecule experiments with Au nanoshell dimers show caspase-3 activation

Subtilisin-like proteases of malaria parasite Plasmodium falciparum (PfSUB1, 2 and 3) are expressed at late asexual blood stages. PfSUB1 and 2 are considered important drug targets due to their essentiality for parasite blood stages and role in merozoite egress and invasion of erythrocytes. We have earlier shown the in vitro serine protease activity of PfSUB3 and its localization at asexual blood stages. In this study, we attempted to identify the biological substrate(s) of PfSUB3 and found parasite profilin (PfPRF) as a substrate of the protease. Eukaryotic profilins are multifunctional proteins with primary role in regulation of actin filament assembly. PfPRF possesses biochemical features of eukaryotic profilins and its rodent ortholog is essential in blood stages. Profilin from related apicomplexan parasite Toxoplasma gondii (TgPRF) is known to be involved in parasite motility, host cell invasion, active egress from host cell, immune evasion and virulence in mice. In this study, mature PfSUB3 proteolysed recombinant PfPRF in a dose-dependent manner in in vitro assays. Recombinant PfPRF was assessed for its proinflammatory activity and found to induce high level of TNF-α and low but significant level of IL-12 from mouse bone marrow-derived dendritic cells. Proteolysis of PfPRF by PfSUB3 is suggestive of the probable role of the protease in the processes of motility, virulence and immune evasion.

The present investigation examined the effects of supplementation of milk peptide fractions produced by enzymatic hydrolysis on the fermentation of reconstituted skim milk (RSM). Changes in pH, cell growth, proteolyticactivity, and angiotensin-converting enzyme (ACE)-inhibitory activity were monitored during fermentation of RSM by pure cultures of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus. The study showed that supplementation with peptide fractions of different molecular weights did not significantly affect the bacterial growth in RSM. All bacteria showed an increased proteolyticactivity in RSM supplemented with large peptides (>10 kDa), and L. helveticus in general exhibited the highest proteolyticactivity among the bacteria studied. The ACE-inhibitory activity was observed to be the maximum in RSM supplemented with larger peptides (>10 kDa) for all bacteria. The results suggest that proteolysis by bacteria leads to increased production of ACE-inhibitory peptides compared to the supplemented peptides produced by enzymatic hydrolysis.

Learning and memory require alteration in number and strength of existing synaptic connections. Extracellular proteolysis within the synapses has been shown to play a pivotal role in synaptic plasticity by determining synapse structure, function, and number. Although synaptic plasticity of excitatory synapses is generally acknowledged to play a crucial role in formation of memory traces, some components of neural plasticity are reflected by nonsynaptic changes. Since information in neural networks is ultimately conveyed with action potentials, scaling of neuronal excitability could significantly enhance or dampen the outcome of dendritic integration, boost neuronal information storage capacity and ultimately learning. However, the underlying mechanism is poorly understood. With this regard, several lines of evidence and our most recent study support a view that activity of extracellular proteases might affect information processing in neuronal networks by affecting targets beyond synapses. Here, we review the most recent studies addressing the impact of extracellular proteolysis on plasticity of neuronal excitability and discuss how enzymatic activity may alter input-output/transfer function of neurons, supporting cognitive processes. Interestingly, extracellular proteolysis may alter intrinsic neuronal excitability and excitation/inhibition balance both rapidly (time of minutes to hours) and in long-term window. Moreover, it appears that by cleavage of extracellular matrix (ECM) constituents, proteases may modulate function of ion channels or alter inhibitory drive and hence facilitate active participation of dendrites and axon initial segments (AISs) in adjusting neuronal input/output function. Altogether, a picture emerges whereby both rapid and long-term extracellular proteolysis may influence some aspects of information processing in neurons, such as initiation of action potential, spike frequency adaptation, properties of action potential and dendritic

Matrix metalloproteinase (MMP) expression and production are associated with advanced-stage tumor and contribute to tumor progression, invasion and metastases. The current study was designed to determine the expression and production of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) by human lymphoid tumor cells. Changes in expression and production were also investigated during tumor progression of multiple myeloma and mycosis fungoides. In situ hybridization analysis revealed that lymphoblastic leukemia B cells (SB cell line), multiple myeloma (MM) cells (U266 cell line) and lymphoblastic leukemia T cells (CEM and Jurkat cell lines) express constitutively the mRNA for MMP-2 and/or MMP-9. We demonstrated by gelatin-zymography of cell culture medium that both enzymes were secreted in their cleaved (activated) form. In situ hybridization of bone marrow plasma cells and gelatin- zymography of the medium showed that patients with active MM (diagnosis, relapse, leukemic progression) express higher levels of MMP-2 mRNA and protein than patients with non-active MM (complete/objective response, plateau) and with monoclonal gammopathies of undetermined significance (MGUS). MMP-9 expression and secretion was similar in all patient groups. In patients with mycosis fungoides (MF), the expression of MMP-2 and MMP-9 mRNAs was significantly upregulated with advancing stage, in terms of lesions both positive for one of two mRNAs and with the greatest intensity of expression. Besides MF cells, the MMP-2 and/or MMP-9 mRNAs were expressed by some stromal cell populations (microvascular endothelial cells, fibroblasts, macrophages), suggesting that these cells cooperate in the process of tumor invasion. Our studies identify MMPs as an important class of proteinases involved in the extracellular matrix (ECM) degradation by human lymphoid tumors, and suggest that MMPs inhibitors may lead to important new treatment for their control. PMID:11097203

Enterokinase is a serine protease of the duodenal brush border membrane that cleaves trypsinogen and produces active trypsin, thereby leading to the activation of many pancreatic digestive enzymes. Overlapping cDNA clones that encode the complete human enterokinase amino acid sequence were isolated from a human intestine cDNA library. Starting from the first ATG codon, the composite 3696 nt cDNA sequence contains an open reading frame of 3057 nt that encodes a 784 amino acid heavy chain followed by a 235 amino acid light chain; the two chains are linked by at least one disulfide bond. The heavy chain contains a potential N-terminal myristoylation site, a potential signal anchor sequence near the amino terminus, and six structural motifs that are found in otherwise unrelated proteins. These domains resemble motifs of the LDL receptor (two copies), complement component Clr (two copies), the metalloprotease meprin (one copy), and the macrophage scavenger receptor (one copy). The enterokinase light chain is homologous to the trypsin-like serine proteinases. These structural features are conserved among human, bovine, and porcine enterokinase. By Northern blotting, a 4.4 kb enterokinase mRNA was detected only in small intestine. The enterokinase gene was localized to human chromosome 21q21 by fluorescence in situ hybridization.

Cell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple-helices occurs within blades I and II of this domain. Examination of simultaneous membrane interaction and triple-helix binding revealed a possible regulation of proteolysis due to steric effects of the membrane. At bicelle concentrations of 1%, enzymatic activity towards triple-helices was increased 1.5-fold. A single mutation in the putative membrane interaction region of MT1-MMP (Ser466Pro) resulted in lower enzyme activation by bicelles. An initial structural framework has thus been developed to define the role(s) of cell membranes in modulating proteolysis. PMID:27405411

Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.

Osteoarthritis and rheumatoid arthritis are destructive joint diseases that involve the loss of articular cartilage. Degradation of cartilage extracellular matrix is believed to occur due to imbalance between the catabolic and anabolic processes of resident chondrocytes. Previous work has suggested that various lysosomal cysteine cathepsins participate in cartilage degeneration; however, their exact roles in disease development and progression have not been elucidated. In order to study degradation processes under conditions resembling the in vivo milieu of the cartilage, we cultivated chondrocytes on a type II collagen-containing matrix. Stimulation of the cultivated chondrocytes with interleukin-1α and/or tumor necrosis factor α resulted in a time-dependent increase in cathepsin S expression and induced its secretion into the conditioned media. Using a novel bioluminescent activity-based probe, we were able to demonstrate a significant increase in proteolyticactivity of cathepsin S in the conditioned media of proinflammatory cytokine-stimulated chondrocytes. For the first time, cathepsin S was demonstrated to be secreted from chondrocytes upon stimulation with the proinflammatory cytokines, and displayed proteolyticactivity in culture supernatants. Its stability at neutral pH and potent proteolyticactivity on extracellular matrix components mean that cathepsin S may contribute significantly to cartilage degradation and may thus be considered a potential drug target in joint diseases.

Chemerin is a chemoattractant involved in innate and adaptive immunity as well as an adipokine implicated in adipocyte differentiation. Chemerin circulates as an inactive precursor in blood whose bioactivity is closely regulated through proteolytic processing at its C terminus. We developed methodology for production of different recombinant chemerin isoforms (chem163S, chem157S, and chem155A) which allowed us to obtain large quantities of these proteins with purity of >95%. Chem158K was generated from chem163S by plasmin cleavage. Characterization by mass spectrometry and Edman degradation demonstrated that both the N and C termini were correct for each isoform. Ca(2+) mobilization assays showed that the EC(50) values for chem163S and chem158K were 54.2 ± 19.9 nm and 65.2 ± 13.2 nm, respectively, whereas chem157S had a ∼50-fold higher potency with an EC(50) of 1.2 ± 0.7 nm. Chem155A had no agonist activity and weak antagonist activity, causing a 50% reduction of chem157S activity at a molar ratio of 100:1. Similar results were obtained in a chemotaxis assay. Because chem158K is the dominant form in cerebrospinal fluid from patients with glioblastoma (GBM), we examined the significance of chemerin in GBM biology. In silico analysis showed chemerin mRNA was significantly increased in tissue from grade III and IV gliomas. Furthermore, U-87 MG cells, a human GBM line, express the chemerin receptors, chemokine-like receptor 1 and chemokine receptor-like 2, and chem157S triggered Ca(2+) flux. This study emphasized the necessity of appropriate C-terminal proteolytic processing to generate the likely physiologic form of active chemerin, chem157S, and suggested a possible role in malignant GBM.

Barley (Hordeum vulgare L.) cysteine proteases are of fundamental biological importance during germination but may also have a large potential as commercial enzyme. Barley cysteine endoprotease B2 (HvEPB2) was expressed in Pichia pastoris from a pPICZαA based construct encoding a HvEPB2 C-terminal truncated version (HvEPB2ΔC) and a proteolytic resistant His6 tag. Maximum yield was obtained after 4 days of induction. Recombinant HvEPB2ΔC (r-HvEPB2ΔC) was purified using a single step of Ni(2+)-affinity chromatography. Purified protein was evaluated by SDS-PAGE, Western blotting and activity assays. A purification yield of 4.26 mg r-HvEPB2ΔC per L supernatant was obtained. r-HvEPB2ΔC follows first order kinetics (Km=12.37 μM) for the substrate Z-Phe-Arg-pNA and the activity was significantly inhibited by the cysteine protease specific inhibitors E64 and leupeptin. The temperature optimum for r-HvEPB2ΔC was 60°C, thermal stability T50 value was 44°C and the pH optimum was 4.5. r-HvEPB2ΔC was incubated with native purified barley seed storage proteins for up to 48 h. After 12h, r-HvEPB2ΔC efficiently reduced the C and D hordeins almost completely, as evaluated by SDS-PAGE. The intensities of the B and γ hordein bands decreased continuously over the 48 h. No degradation occurred in the presence of E64. Recombinant hordeins (B1, B3 and γ1) were expressed in Escherichia coli. After 2h of incubation with r-HvEPB2ΔC, an almost complete degradation of γ1 and partial digests of hordein B1 and B3 were observed.

Panretinal photocoagulation (PRP) reduces the incidence of severe visual loss in proliferative diabetic retinopathy (PDR). The aim of the study was to determine the effect of PRP on the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9, and also on the alpha(2)-Macroglobulin (alpha(2)M) proteolytic state in the vitreous of eyes with PDR. Vitreous samples were obtained from patients undergoing vitrectomy for the treatment of retinal diseases: 17 with PDR and eight with idiopathic macular hole (MH). Qualitative evaluation of the MMP-2 and MMP-9 activation status was performed by gelatin zymography and quantitative assay was carried out for vitreous total protein content and alpha(2)M. The proteolytic state of alpha(2)M was evaluated by Western blotting. Although all vitreous samples contained proMMP-2, increased proMMP-9 and active MMP-9 were detected in PDR samples without PRP. In addition, after PRP the proMMP-9 activity was significantly decreased, whereas the proMMP-2 activity was not affected. Enhanced total protein and alpha(2)M concentrations were observed in all vitreous samples from PDR patients with and without previous PRP compared with samples from patients with MH. However, a differential proteolytic state of alpha(2)M, expressed as 180/85-90kDa ratio, was detected among PDR patients with and without PRP treatment. Whereas a low 180/85-90kDa ratio of alpha(2)M in vitreous of PDR patients without PRP was observed, a high proportion of 180kDa subunit was principally detected in PDR with PRP. These results demonstrate that PDR occurs with an enhanced activity of MMP-9 and activation of alpha(2)M by proteinases, which is reversed after PRP. In addition, we suggest that alpha(2)M plays a key role in the control and regulation of the ocular neovascularization involved in the pathogenesis of ischemic retinal diseases such as PDR.

Proteolytic processing of human host cell factor 1 (HCF-1) to its mature form was recently shown, unexpectedly, to occur in a UDP-GlcNAc-dependent fashion within the transferase active site of O-GlcNAc-transferase (OGT) (Lazarus, M. B., Jiang, J., Kapuria, V., Bhuiyan, T., Janetzko, J., Zandberg, W. F., Vocadlo, D. J., Herr, W., and Walker, S. (2013) Science 342, 1235–1239). An interesting mechanism involving formation and then intramolecular rearrangement of a covalent glycosyl ester adduct of the HCF-1 polypeptide was proposed to account for this unprecedented proteolyticactivity. However, the key intermediate remained hypothetical. Here, using a model enzyme system for which the formation of a glycosyl ester within the enzyme active site has been shown unequivocally, we show that ester formation can indeed lead to proteolysis of the adjacent peptide bond, thereby providing substantive support for the mechanism of HCF-1 processing proposed. PMID:26515062

Caspase 10 (CsCasp10) was identified from a constructed cDNA library of freshwater murrel (otherwise called snakehead) Channa striatus. The CsCasp10 is 1838 base pairs (bp) in length and it is encoding 549 amino acid (aa) residues. CsCasp10 amino acid contains two death effector domains (DED) in the N-terminal at 2-77 and 87-154 and it contains caspase family p20 domain (large subunit) and caspase family p10 domain (small subunit) in the C-terminal at 299-425 and 449-536 respectively. Pairwise analysis of CsCasp10 showed the highest sequence similarity (79%) with caspase 10 of Paralichthys olivaceus. Moreover, the phylogenetic analysis showed that CsCasp10 is clustered together with other fish caspase 10, formed a sister group with caspase 10 from other lower vertebrates including amphibian, reptile and birds and finally clustered together with higher vertebrates such as mammals. Significantly (P < 0.05) highest CsCasp10 gene expression was noticed in gills and lowest in intestine. Furthermore, the CsCasp10 gene expression in C. striatus was up-regulated in gills by fungus Aphanomyces invadans and bacteria Aeromonas hydrophila induction. The proteolyticactivity was analyzed using the purified recombinant CsCasp10 protein. The results showed the proteolyticactivity of CsCasp10 for caspase 10 substrate was 2.5 units per μg protein. Moreover, the proteolyticactivities of CsCasp10 in kidney and spleen induced by A. invadans and A. hydrophila stimulation were analyzed by caspase 10 activity assay kit. All these results showed that CsCasp10 are participated in immunity of C. striatus against A. invadans and A. hydrophila infection.

Exported proteases of Helicobacter pylori (H. pylori) are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI). Among these, we found the predicted serine protease HtrA (Hp1019), which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolyticactivity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolyticactivity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies. PMID:18946507

Exported proteases of Helicobacter pylori (H. pylori) are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI). Among these, we found the predicted serine protease HtrA (Hp1019), which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolyticactivity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolyticactivity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies.

As part of a screening of latex endopeptidases from plants growing in Argentina, the presence of proteolyticactivity in the latex of Funastrum clausum stems is reported. The proteases present in the crude extract showed the main characteristics of the cysteine proteolytic class, i.e. optimum pH at alkaline range, isoelectric point (pI) higher than 9.0, and inhibition of proteolyticactivity by thiol blocking reagents. A remarkable thermal stability was also evident in the crude extract. Endosterolytic preference tried on p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids was higher for the alanine, asparagine and tyrosine derivatives. Preliminary peptidase purification by two-step ionic exchange showed the presence of two proteolytic fractions with molecular masses of approximately 24.0 kDa according to SDS-PAGE.

Microbial infection leads to proteolyticactivation of Drosophila spätzle, which binds to the toll receptor and induces the synthesis of immune proteins. To test whether or not this mechanism exists in lepidopteran insects, we cloned the cDNA of Bombyx mori spätzle-1 and overexpressed the full-length and truncated BmSpz1 cDNA in Escherichia coli. The insoluble fusion proteins were affinity-purified under denaturing condition. After the silkworm larvae were injected with renatured BmSpz1, mRNA levels of antimicrobial peptide genes greatly increased. Similar transcriptional up-regulation was also found in Manduca sexta. Injection of pro-BmSpz1 had no such effect. When pro-BmSpz1 and Micrococcus luteus were incubated with the plasma from M. sexta larvae, we detected proteolytic processing of pro-BmSpz1. These results suggest that active spätzle is required for the induced production of antimicrobial peptides in B. mori and M. sexta. PMID:17337053

Chikungunya virus (CHIKV) capsid protein (CVCP) is a serine protease that possesses cis-proteolyticactivity essential for the structural polyprotein processing and plays a key role in the virus life cycle. CHIKV being an emerging arthropod-borne pathogenic virus, is a public health concern worldwide. No vaccines or specific antiviral treatment is currently available for chikungunya disease. Thus, it is important to develop inhibitors against CHIKV enzymes to block key steps in viral reproduction. In view of this, CVCP was produced recombinantly and purified to homogeneity. A fluorescence resonance energy transfer (FRET)-based proteolytic assay was developed for high throughput screening (HTS). A FRET peptide substrate (DABCYL-GAEEWSLAIE-EDANS) derived from the cleavage site present in the structural polyprotein of CVCP was used. The assay with a Z’ factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors. Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 103 M−1 sec−1 respectively. The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV. PMID:26439734

Virtually all strains of the human pathogenic bacterium Streptococcus pyogenes express a highly conserved extracellular cysteine protease. The protein is made as an inactive zymogen of 40,000 Da and undergoes autocatalytic truncation to result in a 28,000-Da active protease. Numerous independent lines of investigation suggest that this enzyme participates in one or more phases of host-parasite interaction, such as inflammation and soft tissue invasion. Replacement of the single cysteine residue (C-192) with serine (C192S mutation) resulted in loss of detectable proteolyticactivity against bovine casein, human fibronectin, and the low-molecular-weight synthetic substrate 7-amino-4-trifluoromethyl coumarin. The C192S mutant molecule does not undergo autocatalytic processing of zymogen to mature form. Taken together, these data support the hypothesis that C-192 participates in active-site formation and enzyme catalysis. PMID:8675287

Virtually all strains of the human pathogenic bacterium Streptococcus pyogenes express a highly conserved extracellular cysteine protease. The protein is made as an inactive zymogen of 40,000 Da and undergoes autocatalytic truncation to result in a 28,000-Da active protease. Numerous independent lines of investigation suggest that this enzyme participates in one or more phases of host-parasite interaction, such as inflammation and soft tissue invasion. Replacement of the single cysteine residue (C-192) with serine (C192S mutation) resulted in loss of detectable proteolyticactivity against bovine casein, human fibronectin, and the low-molecular-weight synthetic substrate 7-amino-4-trifluoromethyl coumarin. The C192S mutant molecule does not undergo autocatalytic processing of zymogen to mature form. Taken together, these data support the hypothesis that C-192 participates in active-site formation and enzyme catalysis.

The proteolyticactivity of 11 treponemal strains representing different phylogenetic groups was investigated by SDS-polyacrylamide gel electrophoresis with copolymerised casein, gelatin and fibrinogen as substrates. The activity was specified to be trypsin- and chymotrypsin-like by the cleavage of synthetic substrates BAPNA and SAAPFNA, respectively. Nine strains degrade casein and the synthetic substrate BAPNA. Chymotrypsin-like activity specifically inhibited by phenylmethylsulfonyl fluoride was found in four treponemes. Southern blot analysis using a Treponema socranskii subsp. socranskii-specific prtP probe confirmed the presence of prtP homologous genes in these four strains. The internal fragments of the chymotrypsin-like protease genes were cloned and sequenced after PCR amplification. Here we report the cloning of the complete prtP-like gene of T. socranskii subsp. socranskii, an organism shown to possess epidemiologic relevance in periodontitis.

There is an increasing demand of functional foods in developed countries. Yogurt plays an important role in the management of blood pressure. Several bioactive peptides isolated from Allium sativum or fish collagen have shown antihypertensive activity. Thus, in the present study the effects of A. sativum and/or Fish Collagen (FC) on proteolysis and ACE inhibitory activity in yogurt (0, 7 and 14 day) and cheese (0, 14 and 28 day) were investigated. Proteolyticactivities were the highest on day 7 of refrigerated storage in A. sativum-FC-yogurt (337.0 +/- 5.3 microg g(-1)) followed by FC-yogurt (275.3 +/- 2.0 microg g(-1)), A. sativum-yogurt (245.8 +/- 4.2 microg g(-1)) and plain-yogurt (40.4 +/- 1.2 microg g(-1)). On the other hand, proteolyticactivities in cheese ripening were the highest (p < 0.05) on day 14 of storage for plain and A. sativum-cheeses (411.4 +/- 4.3 and 528.7 +/- 1.6 microg g(-1), respectively). However, the presence of FC increased the proteolysis to the highest level on day 28 of storage for FC- and A. sativum-FC cheeses (641.2 +/- 0.1 and 1128.4 +/- 4.5 microg g(-1), respectively). In addition, plain- and A. sativum-yogurts with or without FC showed maximal inhibition of ACE on day 7 of storage. Fresh plain- and A. sativum-cheeses showed ACE inhibition (72.3 +/- 7.8 and 50.4 +/- 1.6 % respectively), the presence of FC in both type of cheeses reduced the ACE inhibition to 62.9 +/- 0.8 and 44.5 +/- 5.0%, respectively. However, refrigerated storage increased ACE inhibition in cheeses (p < 0.05 on day 28) in the presence of FC more than in the absence. In conclusion, the presence of FC in A. sativum-yogurt or cheese enhanced the proteolyticactivity. Thus, it has potential in the development of an effective dietary strategy for hypertension associated cardiovascular diseases.

In hyperinsulinemic states including obesity and T2DM, overproduction of fatty acid and triglyceride contributes to steatosis of the liver, hyperlipidemia and hepatic insulin resistance. This effect is mediated in part by the transcriptional regulator sterol responsive element binding protein-1c (SREBP-1c), which stimulates the expression of genes involved in hepatic fatty acid and triglyceride synthesis. SREBP-1c is up regulated by insulin both via increased transcription of nascent full-length SREBP-1c and by enhanced proteolytic processing of the endoplasmic reticulum (ER)-bound precursor to yield the transcriptionally active n-terminal form, nSREBP-1c. Polyunsaturated fatty acids of marine origin (n-3 PUFA) prevent induction of SREBP-1c by insulin thereby reducing plasma and hepatic triglycerides. Despite widespread use of n-3 PUFA supplements to reduce triglycerides in clinical practice, the exact mechanisms underlying their hypotriglyceridemic effect remain elusive. Here we demonstrate that the n-3 PUFA docosahexaenoic acid (DHA; 22:5 n-3) reduces nSREBP-1c by inhibiting regulated intramembrane proteolysis (RIP) of the nascent SREBP-1c. We further show that this effect of DHA is mediated both via activation of AMP-activated protein kinase (AMPK) and by inhibition of mechanistic target of rapamycin complex 1 (mTORC1). The inhibitory effect of AMPK on SREBP-1c processing is linked to phosphorylation of serine 365 of SREBP-1c in the rat. We have defined a novel regulatory mechanism by which n-3 PUFA inhibit induction of SREBP-1c by insulin. These findings identify AMPK as an important negative regulator of hepatic lipid synthesis and as a potential therapeutic target for hyperlipidemia in obesity and T2DM.

Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolyticactivity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolyticactivity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolyticactivity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors

Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolyticactivity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolyticactivity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolyticactivity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors

Angelman syndrome, a severe neurodevelopmental disease, occurs primarily due to genetic defects, which cause lack of expression or mutations in the wild-type E6AP/UBE3A protein. A proportion of the Angelman syndrome patients bear UBE3A point mutations, which do not interfere with the expression of the full-length protein, however, these individuals still develop physiological conditions of the disease. Interestingly, most of these mutations are catalytically defective, thereby indicating the importance of UBE3A enzymatic activity role in the Angelman syndrome pathology. In this study, we show that Angelman syndrome-associated mutants interact strongly with the proteasome via the S5a proteasomal subunit, resulting in an overall inhibitory effect on the proteolyticactivity of the proteasome. Our results suggest that mutated catalytically inactive forms of UBE3A may cause defects in overall proteasome function, which could have an important role in the Angelman syndrome pathology.

Black skin is more resistant to the deleterious effects of ultraviolet radiation than white skin. A higher melanin content and a different melanosomal dispersion pattern in the epidermis are thought to be responsible for this. Our purpose was to compare skin responses in black and white skin following exposure to solar-simulating radiation (SSR) to further investigate the photoprotective properties of black skin. Six volunteers of skin phototype I-III (white) were exposed to (doses measured directly with a Waldmann UV detector device) 12,000-18,000 mJ per cm2 (2 MED) of SSR and compared with six volunteers of skin phototype VI (black) exposed to 18,000 mJ per cm2 (<1 MED) of SSR. The presence and distribution of skin pigment, DNA photodamage, infiltrating neutrophils, photoaging associated proteolytic enzymes, keratinocyte activation, and the source of interleukin 10 (IL-10) in skin biopsies taken before and after exposure were studied. In all white skinned subjects, 12,000-18,000 mJ per cm2 of SSR induced DNA damage in epidermal and dermal cells, an influx of neutrophils, activeproteolytic enzymes, and diffuse keratinocyte activation. Additionally, in three of the white skinned volunteers IL-10 positive neutrophils were found to infiltrate the epidermis. Except for DNA damage in the supra basal epidermis, none of these changes was found in black skinned subjects. Increased skin pigmentation appears to be primarily responsible for the observed differences in skin responses. Our data could provide an explanation as to why black skin is less susceptible to sunburn, photoaging, and skin carcinogenesis.

A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Kallikrein-related peptidase-8 (KLK8) is a relatively uncharacterized epidermal protease. Although proposed to regulate skin-barrier desquamation and recovery, the catalytic activity of KLK8 was never demonstrated in human epidermis, and its regulators and targets remain unknown. Herein, we elucidated for the first time KLK8 activity in human non-palmoplantar stratum corneum and sweat ex vivo. The majority of stratum corneum and sweat KLK8 was catalytically active, displaying optimal activity at pH 8.5 and considerable activity at pH 5. We also showed that KLK8 is a keratinocyte-specific protease, not secreted by human melanocytes or dermal fibroblasts. KLK8 secretion increased significantly upon calcium induction of terminal keratinocyte differentiation, suggesting an active role for this protease in upper epidermis. Potential activators, regulators, and targets of KLK8 activity were identified by in vitro kinetic assays using pro-KLK8 and mature KLK8 recombinant proteins produced in Pichia pastoris. Mature KLK8 activity was enhanced by calcium and magnesium ions and attenuated by zinc ions and by autocleavage after Arg164. Upon screening KLK8 cleavage of a library of FRET-quenched peptides, trypsin-like specificity was observed with the highest preference for (R/K)(S/T)(A/V) at P1-P1′-P2′. We also demonstrated that KLK5 and lysyl endopeptidase activate latent pro-KLK8, whereas active KLK8 targets pro-KLK11, pro-KLK1, and LL-37 antimicrobial peptide activation in vitro. Together, our data identify KLK8 as a new active serine protease in human stratum corneum and sweat, and we propose regulators and targets that augment its involvement in a skin barrier proteolytic cascade. The implications of KLK8 elevation and hyperactivity in desquamatory and inflammatory skin disease conditions remain to be studied. PMID:20940292

The development of stable enzymes is a key issue in both the food and feed industries. Consequently, the aim of the current study is to evaluate the impact of various additives (sodium chloride, sodium citrate, mannitol, methylparaben, polyethylene glycol 3350, ethylenediaminetetraacetic acid disodium salt, and a serine protease inhibitor) on the stability of a mushroom phytase produced by solid-state cultivation and recovery. Also observed was the effect of the additives on microbial growth inhibition by monitoring both the change in optical density over 30 days of storage and proteolyticactivity. Initially, eight experimental formulations were prepared along with a control. After screening, a 3(2) factorial design was applied to define suitable concentrations of the selected additives. Among the eight formulations tested, the formulation containing NaCl, PEG 3350, and methylparaben retained all of the initial phytase activity after 50 days of storage, with no detected interference from protease activity. Sodium citrate, a metal chelation agent, presented the unusual effect of reducing protease activity in the formulations. Although all formulations presented better phytase stability when compared to the control, NaCl and PEG were both able to prolong the stability of the enzyme activity and also to inhibit microbial growth during storage, making them favorable for application as food and feed additives.

We investigated whether betulin affects the gene expression, secretion and proteolyticactivity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective effect of betulin. Rabbit articular chondrocytes were cultured and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of betulin on IL-1β-induced secretion and proteolyticactivity of MMP-3 was investigated using western blot analysis and casein zymography, respectively. Effect of betulin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) betulin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) betulin inhibited the secretion and proteolyticactivity of MMP-3; (3) betulin suppressed the production of MMP-3 protein in vivo. These results suggest that betulin can regulate the gene expression, secretion, and proteolyticactivity of MMP-3, by directly acting on articular chondrocytes. PMID:28066137

We investigated whether betulin affects the gene expression, secretion and proteolyticactivity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective effect of betulin. Rabbit articular chondrocytes were cultured and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of betulin on IL-1β-induced secretion and proteolyticactivity of MMP-3 was investigated using western blot analysis and casein zymography, respectively. Effect of betulin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) betulin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) betulin inhibited the secretion and proteolyticactivity of MMP-3; (3) betulin suppressed the production of MMP-3 protein in vivo. These results suggest that betulin can regulate the gene expression, secretion, and proteolyticactivity of MMP-3, by directly acting on articular chondrocytes.

The proteolyticactivity of the neutrophil serine-proteinase cathepsin G (CG) on platelet adherence receptors, the glycoprotein (GP) Ib-IX complex and the integrin alpha IIb beta 3, has been investigated. In the range 50 to 200 nmol/l, CG is a potent platelet agonist which induces shape change, granule exocytosis and aggregation. Investigation of the proteolysis of the receptors' subunits during the course of platelet activation by CG was performed by immunoblot analysis of platelet proteins using a panel of specific antibodies. Exposure of platelets for 3 min at 37 degrees C to CG at a concentration that induces full cell activation resulted in an extensive cleavage of the N-terminal region of the extracellular domain of GPIb alpha, the largest (relative molecular mass, M(r), 143,000) of the three subunits constituting the GPIb-IX complex. In contrast, no detectable proteolytic modification of the two other subunits, GPIb beta and GPIX, was detected. Similarly, we observed that neither of the two subunits of the alpha IIb beta 3 receptor were proteolytically modified by CG. Cleavage of GPIb alpha by CG leaves a remnant of the polypeptide chain with M(r) approx. 106,000 in the plasma membrane, while releasing into the extracellular milieu the N-terminal domain with M(r) in the range 40,000 to 46,000. N-terminal sequencing of the CG-derived fragments of GPIb alpha indicated that the Leu275-Tyr276 peptide bond was the primary cleavage site for this proteinase. Proteolysis of GPIb alpha was already detectable at concentrations of CG as low as 25 nmol/l, while with 200 nmol/l the cleavage was detected as soon as 10 s after exposure of platelets to the proteinase. Comparison of the kinetics and concentration dependency for the proteolysis of GPIb alpha and for the activation of platelets by CG showed that cleavage of the GPIb-IX receptor is an early event that accompanies exocytosis and aggregation. Quantitative evaluation of the conversion of GPIb alpha into its

Most cells produce ATP in the mitochondria by oxidative phosphorylation. However, macrophages, which are major players in the innate immune system, use aerobic glycolysis to produce ATP. HIF-1 (hypoxia-inducible factor-1) regulates expression of glycolysis-related genes and maintains macrophage glycolytic activity. However, it is unclear how HIF-1 activity is maintained in macrophages during normoxia. In this study, we found that macrophages lacking membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14), a potent invasion-promoting protease, exhibited considerably lower ATP levels than wild-type cells. HIF-1 was activated by an unanticipated function of MT1-MMP, which led to the stimulation of ATP production via glycolysis. The cytoplasmic tail of MT1-MMP bound to FIH-1 (factor inhibiting HIF-1), which led to the inhibition of the latter by its recently identified inhibitor, Mint3/APBA3. We have thus identified a new function of MT1-MMP to mediate production of ATP so as to support energy-dependent macrophage functions by a previously unknown non-proteolytic mechanism. PMID:20663879

The paper shows the very first results of a feasibility study where the activity of proteolytic enzymes towards dye-labelled artificial substrates immobilized on the surface of planar optical Ta2O5 waveguide was investigated. Within this project, a chromophore label was developed, synthesized and attached to the carboxy-terminus of specific tripeptides. The goal was to develop a highly sensitive optical assay in order to monitor the activity of serine-proteases by cleavage of the amide bond between peptide and chromophore. On the one hand, a strategy was developed to immobilize the labeled tripeptide unto integrated planar waveguides. On the other hand, an instrument, the so-called "chip-reader" was developed to detect the biological process on the surface of the integrated planar optical waveguide. Surface characteristics were analyzed by XPS, TOF-SIMS and contact angle measurements. A comparison between the effectivity of ATR-photometry on chip using TE0 mode and photometry in transmission mode is discussed.

A peptide that exerts antihyperuricemic activity after oral administration was identified from a microbial protease (alcalase) digest of the water extract of shark cartilage by in vivo activity-guided fractionation, using oxonate-induced hyperuricemic rats. Water extract of shark cartilage was first fractionated by preparative ampholine-free isoelectric focusing, followed by preparative reversed-phase liquid chromatography. The antihyperuricemic activity of the alcalse digests of the obtained fractions was evaluated using an animal model. Alcalase digests of the basic and hydrophobic fractions exerted antihyperuricemic activity. A total of 18 peptides were identified in the alcalase digest of the final active fraction. These peptides were chemically synthesized and evaluated for antihyperuricemic activity. Tyr-Leu-Asp-Asn-Tyr and Ser-Pro-Pro-Tyr-Trp-Pro-Tyr lowered the serum uric acid level via intravenous injection at 5 mg/kg of body weight. Furthermore, orally administered Tyr-Leu-Asp-Asn-Tyr showed antihyperuricemic activity. Therefore, these peptides are at least partially responsible for the antihyperuricemic activity of the alcalase digest of shark cartilage.

The synthesis of bioinspired unnatural backbones leading to foldamers can provide effective peptide mimics with improved properties in a physiological environment. This approach has been applied to the design of structural mimics of membrane active antimicrobial peptides (AMPs) for which activities in vitro have been reported. Yet activities and pharmacokinetic properties in vivo in animal models have remained largely unexplored. Here, we report helical oligourea AMP mimics that are active in vitro against bacterial forms of Bacillus anthracis encountered in vivo, as well as in vivo in inhalational and cutaneous mouse models of B. anthracis infection. The pharmacokinetic profile and the tissue distribution were investigated by β-radio imager whole-body mapping in mice. Low excretion and recovery of the native oligourea in the kidney following intravenous injection is consistent with high stability in vivo. Overall these results provide useful information that support future biomedical development of urea-based foldamer peptide mimics.

PA28, a protein activator of the 20 S proteasome, was previously identified in soluble extracts of bovine red blood cells (Ma, C.-P., Slaughter, C. A., and DeMartino, G. N. (1992) J. Biol. Chem. 267, 10515-10523). To determine whether this regulatory protein is as widely distributed as the proteasome, PA28 content and activity were examined in various eukaryotic tissues by immunoblot analysis and by functional assays of tissue extracts. PA28 protein was present in all sources examined. PA28 activity, however, was not detected in many of these sources, including those with the highest level of PA28 protein. To determine the biochemical basis of this result, PA28 was purified from extracts of rat liver, which had high levels of PA28 protein but no PA28 activity. The resulting purified PA28 had no detectable activity but had native and subunit molecular weights indistinguishable from the active PA28 of bovine red blood cells. Using the inactivation of purified PA28 as an assay, a protein that inactivated PA28 without altering its apparent molecular weight on SDS-polyacrylamide gel electrophoresis was identified, purified, and characterized from bovine liver. It had biochemical and catalytic characteristics similar to those of lysosomal carboxypeptidase B. When leupeptin, an inhibitor of lysosomal carboxypeptidase B, was included in the buffers used for the preparation of PA28, PA28 activity was detected in tissues which otherwise failed to demonstrate this activity. A similar result was obtained when extracts were prepared in a manner that minimized disruption of lysosomes. Other carboxypeptidases such as carboxypeptidase Y and pancreatic carboxypeptidase B also inactivated PA28 without altering its apparent molecular weight. Active PA28 binds to the proteasome to form a protease-activator complex that can be isolated after velocity sedimentation centrifugation through glycerol density gradients. Carboxypeptidase-inactivated PA28 failed to form such a complex

The unique response of desiccation-tolerant, or resurrection plants, to extreme drought is accompanied by major changes in the protein pool, raising the possibility of the involvement of proteases. We detected and characterized proteases present in their active state in leaf extracts of desiccated Ramonda serbica Panč., a resurrection plant from the Balkan Peninsula. Plants desiccated under laboratory conditions and maintained in anhydrobiosis for 4 and 14 months revived upon rehydration. Protease activities were determined spectrophotometrically in solution and by zymography on gels. Several endo- and aminopeptidases were detected and characterized by their pH profiles. Their enzyme class was determined using specific inhibitors. Those with higher activities were a serine endopeptidase active against Bz-Arg-pNA with a pH optimum around 9, and aminopeptidases optimally active at pHs from 7 to 9 against Leu-pNA, Met-pNA, Phe-pNA, Pro-pNA and Ala-pNA. The levels of their activities in leaf extracts from desiccated plants were significantly higher than those from rehydrated plants and from regularly watered plants, implying their involvement in the recovery of vegetative tissues from desiccation.

Yeast cell wall contains a number of proteins that are either non-covalently (Scw-proteins), or covalently (Ccw-proteins) bound to β-1,3-glucan, the latter either through GPI-anchors and β-1,6-glucan, or by alkali labile ester linkages between γ-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins). It was shown that a part of Scw4, previously identified among the non-covalently bound cell wall proteins, was covalently attached to wall polysaccharides by a so far unknown alkali sensitive linkage. Thus Scw4 could be released from cell walls by treatments with hot SDS, mild alkali, or β-1,3-glucanases, respectively. It was further shown that non-covalently bound Scw4 (SDS released) underwent the Kex2 proteolytic processing. In this paper it was demonstrated that Scw4 was also processed by yapsins at a position 9 amino acids downstream of the Kex2 cleavage site. Scw4 cleaved at the yapsin site had a markedly lower potential for covalent attachment to glucan. The overproduction of the fully processed form of Scw4 lead to high mortality, particularly in the stationary phase of growth, and to markedly increased cell size. On the other hand, the overproduction of Scw4 processed only by Kex2 or not processed at all had no apparent change in mortality indicating that only the smallest, completely mature form of Scw4 had the activity leading to observed phenotype changes.

The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to…

Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and accuracy. In this study, we...

Exosomes are small microvesicles secreted from the late endosomal compartment of cells. Although an increasing body of evidence indicates that they play a pivotal role in cell-to-cell communication, the biological functions of exosomes are far from fully understood. Recent work has revealed detailed proteomic profiles of exosomes from cell lines and body fluids, which may provide clues to understanding their biological significance and general importance in human diseases. Metalloproteinases include the cell surface-anchored sheddases a disintegrin and metalloproteinases, as well as cell surface-bound and soluble matrix metalloproteinases and these extracellular proteases have been detected in exosomes by proteomic analyses. Exosomes play a key role in the transfer of proteins to other cells and metalloproteinases may provide a novel platform where ectodomain shedding by these membrane proteases alters the makeup of the recipient cell's surface. This review aims to address some of the facets of exosome biology with particular emphasis on the proteolytic factors and we discuss their potential involvement in human diseases, especially tumor biology.

Ubiquitin is a highly conserved protein involved in several important regulatory processes through its ATP-dependent, covalent ligation to a variety of eukaryotic target proteins. The authors describe here the characterization of ubiquitin conjugation in wheat germ extracts and the subsequent isolation of enzymes involved in conjugation. With {sup 125}I-ubiquitin as a substrate, wheat germ extracts form conjugates with either endogenous or added proteins. Ubiquitin-activating enzyme (E1) was purified from wheat germ extracts by using a modification of the covalent affinity chromatography procedure of Ciechanover et al. E1 from wheat germ, like that from rabbit reticulocytes, formed thiol ester intermediates with ubiquitin in the presence of ATP. Purified E1 preparations contained three polypeptides of apparent molecular masses of 117, 123, and 126 kDa after NaDodSO{sub 4}-PAGE. Under nondenaturing conditions, these proteins have native molecular masses of {approx}115 kDa, indicating that they exist as monomers. They concluded that all three species were E1 on the basis of their coelution with E1 activity, by immunorecognition by anti-human E1 antibodies, and by the similarity of their peptide maps. Furthermore, antibodies prepared against wheat germ E1's recognized E1 from rabbit reticulocytes. All three wheat germ E1's were detected in crude extracts prepared under conditions that minimized proteolysis, suggesting that the heterogeneity of the purified E1 preparations was not the result of posthomogenization breakdown. The immunological similarity of animal and plant E1's indicates that this conjugation enzyme, like ubiquitin, has been conserved through evolution.

Defensins are frontline peptides of mucosal immunity in the animal kingdom, including birds. Their resistance to proteolysis and their ensuing ability to maintain antimicrobial potential remains questionable and was therefore investigated. We have shown by bottom-up mass spectrometry analysis of protein extracts that both avian beta-defensins AvBD2 and AvBD7 were ubiquitously distributed along the chicken gut. Cathepsin B was found by immunoblotting in jejunum, ileum, caecum, and caecal tonsils, while cathepsins K, L, and S were merely identified in caecal tonsils. Hydrolysis product of AvBD2 and AvBD7 incubated with a panel of proteases was analysed by RP-HPLC, mass spectrometry and antimicrobial assays. AvBD2 and AvBD7 were resistant to serine proteases and to cathepsins D and H. Conversely cysteine cathepsins B, K, L, and S degraded AvBD2 and abolished its antibacterial activity. Only cathepsin K cleaved AvBD7 and released Ile4-AvBD7, a N-terminal truncated natural peptidoform of AvBD7 that displayed antibacterial activity. Besides the 3-stranded antiparallel beta-sheet typical of beta-defensins, structural analysis of AvBD7 by two-dimensional NMR spectroscopy highlighted the restricted accessibility of the C-terminus embedded by the N-terminal region and gave a formal evidence of a salt bridge (Asp9-Arg12) that could account for proteolysis resistance. The differential susceptibility of avian defensins to proteolysis opens intriguing questions about a distinctive role in the mucosal immunity against pathogen invasion. PMID:27561012

The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. The role of Ca/sup 2 +/-dependent proteinases in the turn-over of myofibrillar protein in normal anecdysial (intermolt) claw muscle is described. Crab Ca/sup 2 +/-dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca/sup 2 +/-dependent proteinase activity in whole homogenates and 90,000 x g supernatant fractions from muscle homogenates has been characterized with respect to Ca/sup 2 +/ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The specificity of crab Ca/sup 2 +/-dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of /sup 125/I-myosin occurs in two phases, both Ca/sup 2 +/-dependent: (1) heavy chain (M/sub r/ = 200,000) is cleaved into four large fragments (M/sub r/ = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (M/sub r/ = 18,000) is cleaved to a 15,000-Da fragment; (2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca/sup 2 +/-dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca/sup 2 +/-dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy.

The innate immune response of insects includes induced expression of genes encoding a variety of antimicrobial peptides. The signaling pathways that stimulate this gene expression have been well characterized by genetic analysis in Drosophila melanogaster, but are not well understood in most other insect species. One such pathway involves proteolyticactivation of a cytokine called Spätzle, which functions in dorsal-ventral patterning in early embryonic development and in the antimicrobial immune response in larvae and adults. We have investigated the function of Spätzle in a lepidopteran insect, Manduca sexta, in which hemolymph proteinases activated during immune responses have been characterized biochemically. Two cDNA isoforms for M. sexta Spätzle-1 differ due to alternative splicing, resulting in a 10 amino acid residue insertion in the pro-region of proSpätzle-1B that is not present in proSpätzle-1A. The proSpätzle-1A cDNA encodes a 32.7 kDa polypeptide that is 23% and 44% identical to D. melanogaster and Bombyx mori Spätzle-1, respectively. Recombinant proSpätzle-1A was a disulfide-linked homodimer. M. sexta hemolymph proteinase 8 (HP8) cleaved proSpätzle-1A to release Spätzle-C108, a dimer of the carboxyl-terminal 108-residue cystine-knot domain. Injection of Spätzle-C108, but not proSpätzle-1A, into larvae stimulated expression of several antimicrobial peptides and proteins, including attacin-1, cecropin-6, moricin, lysozyme, and the immunoglobulin domain protein hemolin, but did not significantly affect expression of two bacteria-inducible pattern recognition proteins, immulectin-2 and β-1,3-glucan recognition protein-2. Results from this paper and other recent studies support a model for a pathway in which the clip-domain proteinase proHP6 becomes activated in plasma upon exposure to Gram-negative or Gram-positive bacteria or to β-1,3-glucan. HP6 then activates proHP8, which in turn activates Spätzle-1. The resulting Spätzle-C108 dimer is

Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.

The present study describes the in vivo detection and imaging of tumour-associated MMP-7 (matrix metalloproteinase-7 or matrilysin) activity using a novel polymer-based fluorogenic substrate PB-M7VIS, which serves as a selective 'proteolytic beacon' (PB) for this metalloproteinase. PB-M7VIS is built on a PAMAM (polyamido amino) dendrimer core of 14.2 kDa, covalently coupled with an Fl (fluorescein)-labelled peptide Fl(AHX)RPLALWRS(AHX)C (where AHX stands for aminohexanoic acid) and with TMR (tetramethylrhodamine). PB-M7VIS is efficiently and selectively cleaved by MMP-7 with a k (cat)/ K (m) value of 1.9x10(5) M(-1).s(-1) as measured by the rate of increase in Fl fluorescence (up to 17-fold for the cleavage of an optimized PB-M7VIS) with minimal change in the TMR fluorescence. The K (m) value for PB-M7VIS is approx. 0.5 microM, which is approx. two orders of magnitude lower when compared with that for an analogous soluble peptide, indicating efficient interaction of MMP-7 with the synthetic polymeric substrate. With MMP-2 or -3, the k (cat)/ K (m) value for PB-M7VIS is approx. 56- or 13-fold lower respectively, when compared with MMP-7. In PB-M7VIS, Fl(AHX)RPLALWRS(AHX)C is a selective optical sensor of MMP-7 activity and TMR serves to detect both the uncleaved and cleaved reagents. Each of these can be visualized as subcutaneous fluorescent phantoms in a mouse and optically discriminated based on the ratio of green/red (Fl/TMR) fluorescence. The in vivo specificity of PB-M7VIS was tested in a mouse xenograft model. Intravenous administration of PB-M7VIS gave significantly enhanced Fl fluorescence from MMP-7-positive tumours, but not from control tumours ( P <0.0001), both originally derived from SW480 human colon cancer cells. Prior systemic treatment of the tumour-bearing mice with an MMP inhibitor BB-94 ([4-( N -hydroxyamino)-2 R -isobutyl-3 S -(thienylthiomethyl)-succinyl]-L-phenylalanine- N -methylamide), markedly decreased the Fl fluorescence over the MMP-7

Background The herbicides glyphosate (Gly) and imazamox (Imx) inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolyticactivities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling. Results Several common changes in proteolyticactivity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides. Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment. Conclusion These results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolyticactivity remains to be determined. PMID:24040092

Lactobacillus helveticus is one of the species of lactic acid bacteria (LAB) most commonly used in the production of fermented milk beverages and some types of hard cheese. The versatile nature of this bacterium is based on its highly efficient proteolytic system consisting of cell-envelope proteinases (CEPs), transport system and intracellular peptidases. Besides use of L. helveticus in cheese processing, the production of fermented milk preparations with health promoting properties has become an important industrial application. Studies have shown that fermented dairy products are able to decrease blood pressure, stimulate the immune system, promote calcium absorption, and exert an anti-virulent effect against pathogens. These beneficial effects are produced by a variety of peptides released during the hydrolysis of milk proteins by the proteolytic system of L. helveticus, which provides the bacterium with its nutritional requirements for growth. In recent years, studies have focused on understanding the factors that affect the kinetics of milk protein hydrolysis by specific strains and have concentrated on the effect of pH, temperature, growth phase, and matrix composition on the bacterial enzymatic system. This review focuses on the role of the proteolytic system of L. helveticus in the production of bioactive compounds formed during fermentation of dairy products. Taking advantage of the powerful proteolytic system of this bacterium opens up future opportunities to search for novel food-derived compounds with potential health promoting properties. PMID:23467265

Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolyticactivity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolyticactivity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolyticactivity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolyticactivity.

Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolyticactivity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolyticactivity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolyticactivity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolyticactivity. PMID:27283981

Twenty-eight recA mutants, isolated after spontaneous mutagenesis generated by the combined action of RecA1202(Prtc) and UmuDC proteins, were characterized and sequenced. The mutations are intragenic suppressors of the recA1202 allele and were detected by the reduced coprotease activity of the gene product. Twenty distinct mutation sites were found, among which two mutations, recA1620 (V-275-->D) and recA1631 (I-284-->N), were mapped in the C-terminal portion of the interfilament contact region (IFCR) in the RecA crystal. An interaction of this region with the part of the IFCR in which the recA1202 mutation (Q-184-->K) is mapped could occur only intermolecularly. Thus, altered IFCR and the likely resulting change in interfilament association appear to be important aspects of the formation of a constitutively active RecA coprotease. This observation is consistent with the filament-bundle theory (R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 335:318-325, 1992). Furthermore, we found that among the 20 suppressor mutations, 3 missense mutations that lead to recombination-defective (Rec-) phenotypes also mapped in the IFCR, suggesting that the IFCR, with its putative function in interfilament association, is required for the recombinase activity of RecA. We propose that RecA-DNA complexes may form bundles analogous to the RecA bundles (lacking DNA) described by Story et al. and that these RecA-DNA bundles play a role in homologous recombination. Images PMID:8407828

Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH2-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells. PMID:11748167

Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH(2)-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells.

Enterococci show higher proteolyticactivities than other lactic acid bacteria and thus have received considerable attention in scientific literature in recent years. Proteolytic enzymes of enterococci have warranted the use of some species as starter, adjuncts or protective cultures and as probiotics, while in some strains they have also been linked with virulence. Consequently, the isolation and identification of proteolytic enterococci is becoming of increasing interest and importance. However, current screening methods for proteolytic enterococci can be time consuming, requiring a two-step procedure which may take up to 96h. This study describes a method, utilising Kanamycin Skim Milk Aesculin Azide (KSMEA) agar, for the isolation of proteolytic enterococci in one-step, thereby significantly reducing screening time. KSMEA combines the selective properties of Kanamycin Aesculin Azide Agar (KAA) with skim milk powder for the detection of proteolytic enterococci. Enterococci produced colonies with a black halo on KSMEA which were accompanied by a zone of clearing in the media when enterococci were proteolytic. KSMEA medium retained the selectivity of KAA, while proteolytic enterococci were easily distinguished from non-proteolytic enterococci when two known strains were propagated on KSMEA. KSMEA also proved effective at isolating and detecting enterococci in raw milk, faeces and soil. Isolates recovered from the screen were confirmed as enterococci using genus-specific primers. Proteolytic enterococci were present in the raw milk sample only and were easily distinguishable from non-proteolytic enterococci and other microorganisms. Therefore, KSMEA provides a rapid, one-step screening method for the isolation of presumptive proteolytic enterococci.

The gelatinase members of the MMP family have consistently been associated with tumor invasiveness, which make them an attractive target for molecular imaging. We report new activatable proteolytic optical imaging agents that consist of triple-helical peptide (THP) conjugates, with high specificity to the gelatinases, bearing quenched cypate dyes. With quenching efficiencies up to 51%, the amplified fluorescence signal upon cypate3-THP hydrolysis by the gelatinases (kcat/KM values of 6.4 × 103 M−1 s−1 to 9.1 × 103 M−1 s−1 for MMP-2 and MMP-9, respectively) in mice bearing human fibrosarcoma xenografted tumors was monitored with fluorescence molecular tomography. There was significant fluorescence enhancement within the tumor and this enhancement was reduced by treatment with pan-MMP inhibitor, Ilomastat. These data, combined with the gelatinase substrate specificity observed in vitro, indicated the observed fluorescence at the site of the tumor was due to gelatinase mediated hydrolysis of cypate3-THP. PMID:25047578

New magnetic adsorbents for batch isolation and removal of various proteolytic enzymes were prepared by glutaraldehyde cross-linking of bovine, porcine and human erythrocytes in the presence of fine magnetic particles. Trypsin, chymotrypsin, alkaline bacterial protease and proteases present in various commercial enzyme preparations were efficiently adsorbed on these adsorbents; on the contrary, proteins without proteolyticactivity were not adsorbed.

Terasi is a traditional product generally made of fermented shrimp. There were many studies regarding lactic acid bacteria of terasi but none regarding proteolitic bacteria. This study was conducted to isolate and identify the thermophilic and mesophylic proteolytic bacteria from terasi. In addition, the effect of different salt concentrations on the growth of the isolated proteolytic bacteria with the greatest proteolyticactivity was also studied. Terasi samples were obtained from the Northern coast region of Java island i.e. Jepara, Demak and Batang. The study obtained 34 proteolytic isolates. Four isolates were identified as Sulfidobacillus, three isolates as Vibrio / Alkaligenes / Aeromonas, two isolates as Pseudomonas, 21 isolates as Bacillus, three isolates as Kurthia/ Caryophanon and one isolates as Amphibacillus. The growth of proteolytic bacteria was affected by salt concentration. The largest growth was found at 0 ppm salt concentrations and growth was declined as salt concentration increased. Maximum growth at each salt concentration tested was found at 8 hours incubation.

Proteolytic bacteria in paddy field soils under rice cultivation were characterized and enumerated using azocoll agar plates. Bacillus spp. were the proteolytic bacteria that were most frequently present, comprising 59% of the isolates. They were always the numerically dominant proteolytic bacteria isolated from three kinds of fertilizer treatments (yearly application of rice-straw compost and chemical fertilizer, yearly application of chemical fertilizer, and no fertilizer application) and at three different stages of rice development (vegetative growth stage, maximal tillering stage, and harvest stage). Of the 411 proteolytic bacteria isolated, 124 isolates had stronger proteolyticactivity than others on the basis of gelatin liquefaction tests and most of them were Bacillus spp. (100% in 1989 and 92.4% in 1991). Bacillus subtilis and Bacillus cereus were the main bacteria of this group and Bacillus mycoides, Bacillus licheniformis, and Bacillus megaterium were also present. We conclude that these Bacillus spp. are the primary source of soil protease in these paddy fields.

The dependence of the effects of zinc and copper on the activities of proteinases of the stomach and intestinal mucosa on temperature and pH in four species of boreal piscivorous fish (pike Esox lucius, zander Zander lucioperca, perch Perca fluviatilis and burbot Lota lota) as well as in some of their potential preys (kilka Clupeonella cultriventris, ruff Gymnocephalus cernuus, perch and roach Rutilus rutilus) was investigated. Species-specific differences of the effects of these heavy metals upon the activities of proteinases depending on temperature and pH were demonstrated. It was revealed that the stomach mucosa proteinases were more tolerant to the effects of the studied factors than the intestinal mucosa proteinases, especially true for pike. The effects of the heavy metals on the whole body proteinases of the fishes' potential preys were mostly dependent on temperature than on pH. At pH 3.0, the negative action of zinc and copper on the fish digestive tract mucosa proteolyticactivity to a considerable degree was compensated by the high activity of the hemoglobinlytic proteinases, probably, cathepsine D.

Insoluble and fibrillar forms of α-synuclein are the major components of Lewy bodies, a hallmark of several sporadic and inherited neurodegenerative diseases known as synucleinopathies. α-Synuclein is a natural unfolded and aggregation-prone protein that can be degraded by the ubiquitin-proteasomal system and the lysosomal degradation pathways. α-Synuclein is a target of the main cellular proteolytic systems, but it is also able to alter their function further, contributing to the progression of neurodegeneration. Aging, a major risk for synucleinopathies, is associated with a decrease activity of the proteolytic systems, further aggravating this toxic looping cycle. Here, the current literature on the basic aspects of the routes for α-synuclein clearance, as well as the consequences of the proteolytic systems collapse, will be discussed. Finally, particular focus will be given to the sirtuins’s role on proteostasis regulation, since their modulation emerged as a promising therapeutic strategy to rescue cells from α-synuclein toxicity. The controversial reports on the potential role of sirtuins in the degradation of α-synuclein will be discussed. Connection between sirtuins and proteolytic systems is definitely worth of further studies to increase the knowledge that will allow its proper exploration as new avenue to fight synucleinopathies. PMID:25946078

Earlier studies have revealed the ability of sera from several mammals to neutralize the toxic effects of snake venom. The Venezuelan opossum (Didelphis marsupialis) is one that has been found to inhibit hemorrhagic and proteolyticactivities of venoms from many species of snakes. In this article it is shown that the opossum sera and its 0.15DM fraction were able to completely neutralize both hemorrhagic and hydrolysis (proteolysis) of casein effects induced by venom of the Lansberg's hognose pit viper (Porthidium lansbergii hutmanni). We have used DEAE-cellulose ion exchange chromatography to collect protein fractions from D. marsupialis sera which were able to defend mice from the lethal effects of P.l. hutmanni venom. The fractions separated were homogeneous by conventional electrophoresis using SDS-PAGE. The protein bands obtained contained molecular weights of approximately 6 to 220 kDa. These results revealed the presence of proteases inhibitors in the opossum sera fractions and the inhibition of venom activity by opossum sera suggesting a reciprocal adaptation at the molecular level.

Wound healing is a crucial regenerative process in all organisms. We examined expression, integrity, and function of the proteins in the hepatocyte growth factor (HGF)/c-Met signaling pathway in normally healing and non-healing human skin wounds. Whereas in normally healing wounds phosphorylation of c-Met was most prominent in keratinocytes and dermal cells, in non-healing wounds phosphorylation of c-Met was barely detectable, suggesting reduced c-Met activation. In wound exudates obtained from non-healing, but not from healing wounds, HGF protein was a target of substantial proteolytic processing that was different from the classical activation by known serine proteases. Western blot analysis and protease inhibitor studies revealed that HGF is a target of neutrophil elastase and plasma kallikrein during skin repair. Proteolytic processing of HGF by each of these proteases significantly attenuated keratinocyte proliferation, wound closure capacity in vitro, and c-Met signal transduction. Our findings reveal a novel pathway of HGF processing during skin repair. Conditions in which proteases are imbalanced and tend toward increased proteolyticactivity, as in chronic non-healing wounds, might therefore compromise HGF activity due to the inactivation of the HGF protein and/or the generation of HGF fragments that ultimately mediate a dominant negative effect and limit c-Met activation. PMID:19389925

CAPN3/p94/calpain-3, a calpain protease family member predominantly expressed in skeletal muscle, possesses unusually rapid and exhaustive autolytic activity. Mutations in the human CAPN3 gene impairing its protease functions cause limb-girdle muscular dystrophy type 2A (LGMD2A); yet, the connection between CAPN3’s autolytic activity and the enzyme’s function in vivo remain unclear. Here, we demonstrated that CAPN3 protease activity was reconstituted by intermolecular complementation (iMOC) between its two autolytic fragments. Furthermore, the activity of full-length CAPN3 active-site mutants was surprisingly rescued through iMOC with autolytic fragments containing WT amino acid sequences. These results provide evidence that WT CAPN3 can be formed by the iMOC of two different complementary CAPN3 mutants. The finding of iMOC-mediated restoration of calpain activity indicates a novel mechanism for the genotype–phenotype links in LGMD2A. PMID:25512505

The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, in Mycobacterium tuberculosis (Mtb) are essential and, therefore, promising drug targets. The Mtb ClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3–4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity of Mtb ClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2–3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel β-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates. PMID:26858247

Influence of coordinative compounds of germanium (IV) and stanum (IV) (complexes of germanium (IV) with nicotinamide (Nad) [GeCl2(Nad)4]Cl2 (1) and complexes of stanum (IV) with 2-hydroxybenzoilhydrazone 4-dimetylaminobenzaldehide (2-OH-HBdb) [SnCl4(2-OH-Bdb-H)] (2), 3-hydroxy-2-naphtoilhydrazone 2-hydroxynaphtaldehide (3-OH-H2Lnf) [SnCl3(3-OH-HLnf)] (3) and izonicotinoilhydrazone 2-hydroxyibenzaldehide [SnCl3 (Is·H)] (4) on activity of peptidases 1 and 2 Bacillus thuringiensis, α-L-rhamnosidase Cryptococcus albidus, Eupenicillium erubescens and α-amylase Aspergillus flavus var. oryzae. Results testify that all studied compounds differ on their influence on activity of the enzymes tested: significantly don't change elastolytic activity of peptidases 1 and 2 B. thuringiensis, completely inhibit A. flavus var. oryzae amylase, activate or oppress of α-L-rhamnosidase C. albidus and E. erubescens. Considerable differences in compounds (3, 4) on activity observed in case of the last. It's possible that peculiarity of influence (1) in compare with (2-4) is connected with existence of different central atoms of complexants: germanium (IV) (1) and stanum (IV) (2-4). A certain analogy in oppression of C. albidus α-L-rhamnosidase by compounds (1) and (4) can explain with presence of a pyridinic ring at molecules of their ligands. The less activsty displayed compound (2) with coordinative knot {SnCl4ON}. Nature of compounds (3, 4) activity was absolutely different: essential increase of activity of C. albidus α-L-rhamnosidase and full oppression of E. erubescens α-L-rhamnosidase by compound (3), while the action of compound (4) was feed back. Taking into account identical coordination knot {SnCl3O2N} the major role in this case play change of a hydrazide fragment in molecules of their ligands.

Conjugative plasmids and conjugative transposons contain the genes, which products specifically inhibit the type I restriction--modification systems. Here is shown that non-conjugative transposons Tn2l, Tn5053, Tn5045, Tn501, Tn402 partially inhibit the restriction activity of the type I restriction-modification endonuclease EcoKI (R2M2S1) in Escherichia coli cells K12 (the phenomenon of restriction alleviation, RA). Antirestriction activity of the transposons is determined by the MerR and ArdD proteins. Antirestriction activity of transposons is absent in mutants E. coli K12 clpX and clpP and is decreased in mutants E. coli K12 recA, recBC and dnaQ (mutD). Induction of the RA in response to the MerR and ArdD activities is consistent with the production of unmodified target sequences following DNA repair and DNA synthesis associated with recombination repair or replication errors. RA effect is determined by the ClpXP-dependent degradation of the endonuclease activity R subunit of EcoKI (R2M2S1).

Six pepsinogens were purified from the gastric mucosa of largemouth bass (Micropterus salmoides) by DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, and Mono Q FPLC. The potential specific activities of two major pepsinogens, PG1-1 and PG2-2, against hemoglobin were 51 and 118 units/mg protein, respectively. The activity of pepsin 2-2 was the highest among the pepsins reported to date; this might be linked to the strongly carnivorous diet of the largemouth bass. The molecular masses of PG1-1 and PG2-2 were 39.0 and 41.0 kDa, respectively. The N-terminal amino acid sequences of PG1-1 and PG2-2 were LVQVPLEVGQTAREYLE- and LVRLPLIVGKTARQALLE-, respectively, showing similarities with those of fish type-A pepsinogens. The optimal pHs for hemoglobin-digestive activity of pepsins 1-1 and 2-2 were around 1.5 and 2.0, respectively, though both pepsins retained considerable activity at pHs over 3.5. They showed maximal activity around 50 and 40 °C, respectively. They were inhibited by pepstatin similarly to porcine pepsin A. The cleavage specificities clarified with oxidized insulin B chain were shown to be restricted to a few bonds consisting of hydrophobic/aromatic residues, such as the Leu(15)-Tyr(16), Phe(24)-Phe(25) and Phe(25)-Tyr(26) bonds. When hemoglobin was used as a substrate, the kcat/Km value of bass pepsin 2-2 was 4.6- to 36.8-fold larger than those of other fish pepsins. In the case of substance P, an ideal pepsin substrate mimic, the kcat/Km values were about 200-fold larger than those of porcine pepsin A, supporting the high activity of the bass pepsin.

Myostatin, a potent negative regulator of myogenesis, is proteolytically processed by furin proteases into active mature myostatin before secretion from myoblasts. Here, we show that mature myostatin auto-regulates its processing during myogenesis. In a cell culture model of myogenesis, Northern blot analysis revealed no appreciable change in myostatin mRNA levels between proliferating myoblasts and differentiated myotubes. However, Western blot analysis confirmed a relative reduction in myostatin processing and secretion by differentiated myotubes as compared to proliferating myoblasts. Furthermore, in vivo results demonstrate a lower level of myostatin processing during fetal muscle development when compared to postnatal adult muscle. Consequently, high levels of circulatory mature myostatin were detected in postnatal serum, while fetal circulatory myostatin levels were undetectable. Since Furin proteases are important for proteolytically processing members of the TGF-beta superfamily, we therefore investigated the ability of myostatin to control the transcription of furin and auto-regulate the extent of its processing. Transfection experiments indicate that mature myostatin indeed regulates furin protease promoter activity. Based on these results, we propose a mechanism whereby myostatin negatively regulates its proteolytic processing during fetal development, ultimately facilitating the differentiation of myoblasts by controlling both furin protease gene expression and subsequent active concentrations of mature myostatin peptide.

Matrix metalloproteinases (MMP) 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand the molecular role of the gelatinases in tumor development. Recently, a self-assembling homotrimeric triple-helical peptide (THP), incorporating a sequence from type V collagen, with high substrate specificity to the gelatinases has been developed. To determine whether this THP would be suitable for imaging protease activity, 5-carboxyfluorescein (5FAM) was conjugated, resulting in 5FAM3-THP and 5FAM6-THP, which were quenched up to 50%. 5FAM6-THP hydrolysis by MMP-2 and MMP-9 displayed kcat/KM values of 1.5 × 104 and 5.4 × 103 M-1 s-1, respectively. Additionally 5FAM6-THP visualized gelatinase activity in gelatinase positive HT-1080 cells, but not in gelatinase negative MCF-7 cells. Furthermore, the fluorescence in the HT-1080 cells was greatly attenuated by the addition of a MMP-2 and MMP-9 inhibitor, SB-3CT, indicating that the observed fluorescence release was mediated by gelatinase proteolysis and not non-specific proteolysis of the THPs. These results demonstrate that THPs fully substituted with fluorophores maintain their substrate specificity to the gelatinases in human cancer cells and may be useful in in vivo molecular imaging of gelatinase activity.

Matrix metalloproteinases (MMP) 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand the molecular role of the gelatinases in tumor development. Recently, a self-assembling homotrimeric triple-helical peptide (THP), incorporating a sequence from type V collagen, with high substrate specificity to the gelatinases has been developed. To determine whether this THP would be suitable for imaging protease activity, 5-carboxyfluorescein (5FAM) was conjugated, resulting in 5FAM3-THP and 5FAM6-THP, which were quenched up to 50%. 5FAM6-THP hydrolysis by MMP-2 and MMP-9 displayed kcat/KM values of 1.5 × 104 and 5.4 × 103 M−1 s−1, respectively. Additionally 5FAM6-THP visualized gelatinase activity in gelatinase positive HT-1080 cells, but not in gelatinase negative MCF-7 cells. Furthermore, the fluorescence in the HT-1080 cells was greatly attenuated by the addition of a MMP-2 and MMP-9 inhibitor, SB-3CT, indicating that the observed fluorescence release was mediated by gelatinase proteolysis and not non-specific proteolysis of the THPs. These results demonstrate that THPs fully substituted with fluorophores maintain their substrate specificity to the gelatinases in human cancer cells and may be useful in in vivo molecular imaging of gelatinase activity. PMID:24959683

This work discusses the biological and biochemical characterization of an extracellular protease produced by Pseudomonas fluorescens. The enzyme has a molecular weight of 49.486 kDa and hydrolyzes gelatin, casein, and azocasein, but not BSA. Its maximum activity is found at 37°C and pH 7.5, but it retained almost 70% activity at pH 10.0. It was shown to be a metalloprotease inhibited by Cu(2+), Ni(2+), Zn(2+), Hg(2+), Fe(2+), and Mg(2+), but induced by Mn(2+). After incubation at 100°C for 5min, the enzyme presented over 40% activity, but only 14 to 30% when submitted to milder heat treatments. This behavior may cause significant problems under conditions commonly used for the processing and storage of milk and dairy products, particularly UHT milk. A specific peptide sequenced by mass spectrometer analysis allowed the identification of gene that encodes this extracellular protease in the genome of Pseudomonas fluorescens 07A strain. The enzyme has 477 AA and highly conserved Ca(2+)- and Zn(2+)-binding domains, indicating that Ca(2+), the main ion in milk, is also a cofactor. This work contributes to the understanding of the biochemical aspects of enzyme activity and associates them with its sequence and structure. These findings are essential for the full understanding and control of these enzymes and the technological problems they cause in the dairy industry.

Anisi stellati fructus (ASF), commonly known as star anise, has long been used as a traditional Chinese medicine to treat inflammation, nervousness, insomnia and pain. In recent studies, it has been demonstrated that ASF possesses anti-bacterial, anti-fungal and anti-oxidant activities, as well as exhibits inhibitory effects on capillary‑like tube formation in human umbilical vein endothelial cells (HUVECs). However, the effects of ASF extract on the metastatic potential of malignant tumor cells have not been examined. In this study, we found that daily oral administration of ASF (50 mg/kg) remarkably reduced the number of pulmonary metastatic colonies of B16F10 cells in C57BL/6J mice with no observed systemic toxicity. In an in vitro system, ASF inhibited metastatic properties, including anchorage‑independent colony formation, migration and invasion. Upon phorbol 12-myristate 13-acetate (PMA) stimulation, the mRNA levels of matrix metalloproteinases (MMPs) -9, -13, -14 and urokinase plasminogen activator (uPA) decreased in a dose-dependent manner with ASF treatment. Gelatinase, type I collagenase, and uPA activities were also suppressed efficiently by ASF treatment. In response to PMA, NF-κB and AP-1 activation as well as p38 phosphorylation, which are crucial for MMP activation, were significantly decreased by ASF. In particular, ASF considerably inhibited tumor-induced HUVEC migration and tube formation and suppressed in vivo tumor-induced angiogenesis via a reduction of pro-angiogenic factors in tumors. These results collectively indicate that ASF might be useful in the management of metastatic malignant tumors.

The influence of X-radiation on activity of lysosomal enzymes (D, L, H cathepsins) in rat spleen tissue and in inoculated rat sarcoma 45 has been investigated. Intact rats and rats with tumors were subjected to whole-body and sarcoma 45 to local irradiation with doses of 0.155 C/kg and 0.31 C/kg in conditions of breathing gas hypoxic mixture containing 90% of nitrogen and 10% of oxygen (GHM-10). The combined exposure to radiation and GHM-10 was shown to produce a certain protective action (e.g. normalized cathepsin activity) in the spleen. In the tumor tissue the protective effect of GHM-10 was absent.

We have purified a cobalamin-binding protein obtained by papain digestion of pig intestine by cobalamin-AH-Sepharose affinity chromatography, with a purification factor of 17,300, a yield of 63% and a cobalamin-binding activity of 11,260 pmol/mg of protein. The protein contained 3.8% carbohydrate and was O- and N-glycosylated. Its molecular mass was 69 kDa on SDS/PAGE and its isoelectric point was 5.1. It had a binding activity for both [57Co]cobalamin and [57Co]cobalamin-intrinsic factor in native PAGE autoradiography and it inhibited the binding of intrinsic factor to the intact intestinal receptor with an IC50 of 49.31 nmol/l in a radioisotope assay. In conclusion, the purified protein shared a binding activity for both cobalamin and intrinsic factor-cobalamin complexes and could correspond to the extracellular domain of the ileal intrinsic factor receptor. PMID:8573109

ABSTRACT Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is necessary for viral activation and infectivity. In humans and mice, members of the type II transmembrane protease family (TTSP), e.g., TMPRSS2, TMPRSS4, and TMPRSS11d (HAT), have been shown to cleave influenza virus HA for viral activation and infectivity in vitro. Recently, we reported that inactivation of a single HA-activating protease gene, Tmprss2, in knockout mice inhibits the spread of H1N1 influenza viruses. However, after infection of Tmprss2 knockout mice with an H3N2 influenza virus, only a slight increase in survival was observed, and mice still lost body weight. In this study, we investigated an additional trypsin-like protease, TMPRSS4. Both TMPRSS2 and TMPRSS4 are expressed in the same cell types of the mouse lung. Deletion of Tmprss4 alone in knockout mice does not protect them from body weight loss and death upon infection with H3N2 influenza virus. In contrast, Tmprss2−/− Tmprss4−/− double-knockout mice showed a remarkably reduced virus spread and lung pathology, in addition to reduced body weight loss and mortality. Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virus in vivo. IMPORTANCE Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality. Due to high variability of the virus genome, resistance to available antiviral drugs is frequently observed, and new targets for treatment of influenza are needed. Host cell factors essential for processing of the virus hemagglutinin represent very suitable drug targets because the virus is dependent on these host factors for replication. We reported previously that Tmprss2-deficient mice are protected against H1N1 virus infections, but only marginal protection against H3N2 virus infections was observed. Here we show that deletion of two host protease genes, Tmprss2 and

A bacterial strain which can be grown in a medium containing organic solvents and can secrete a proteolytic enzyme was isolated and identified as Pseudomonas aeruginosa. The strain was derived by the following two-step procedures: high proteolytic enzyme producers were first isolated by the usual method, and then the organic solvent-tolerant microorganism was selected from these high-rate proteolytic enzyme producers. The proteolyticactivity of the supernatant of the culture was stable in the presence of various organic solvents. The stability of the enzyme in the presence of organic solvents, of which the values of the logarithm of the partition coefficient (log P) were equal to or more than 3.2, was almost the same as that in the absence of organic solvents. It is expected that both the solvent-tolerant microorganism and the solvent-stable enzyme produced by this strain can be used as catalysts for reactions in the presence of organic solvents.

A fundamental component of systems biology, proteolytic cleavage is involved in nearly all aspects of cellular activities: from gene regulation to cell lifecycle regulation. Current sequencing technologies have made it possible to compile large amount of cleavage data and brought greater understanding of the underlying protein interactions. However, the practical impossibility to exhaustively retrieve substrate sequences through experimentation alone has long highlighted the need for efficient computational prediction methods. Such methods must be able to quickly mark substrate candidates and putative cleavage sites for further analysis. Available methods and expected reliability depend heavily on the type and complexity of proteolytic action, as well as the availability of well-labelled experimental data sets: factors varying greatly across enzyme families. For this review, we chose to give a quick overview of the general issues and challenges in cleavage prediction methods followed by a more in-depth presentation of major techniques and implementations, with a focus on two particular families of cysteine proteases: caspases and calpains. Through their respective differences in proteolytic specificity (high for caspases, broader for calpains) and data availability (much lower for calpains), we aimed to illustrate the strengths and limitations of techniques ranging from position-based matrices and decision trees to more flexible machine-learning methods such as hidden Markov models and Support Vector Machines. In addition to a technical overview for each family of algorithms, we tried to provide elements of evaluation and performance comparison across methods.

Venous leg ulcers (VLU) are characterized by sustained proteolytic microenvironment impairing the healing process. Wound fluid (WF) reflect the biomolecular activities occurring within the wound area; however, it is unclear if WF from different healing phases have different proteolytic profiles and how VLU microenvironment affects the wound healing mechanisms. We investigated the proteolytic network of WF from distinct VLU phases, and in WF- and LPS-stimulated THP-1 monocytes treated with glycosaminoglycan sulodexide, a well known therapeutic approach for VLU healing. WF were collected from patients with VLU during inflammatory (Infl) and granulating (Gran) phases. WF and THP-1 supernatants were analyzed for nine matrix metalloproteinases (MMP) and four tissue inhibitors of metalloproteinases (TIMP) by multiplex immunoassays. Our results demonstrated that: 1) WF from Infl VLU contained significantly increased concentrations of MMP-2, MMP-9, MMP-12, TIMP-1, and TIMP-2 compared to Gran WF; 2) WF from Gran VLU showed significantly increased levels of MMP-1, MMP-7, MMP-13, and TIMP-4 compared to Infl WF; 3) LPS- and WF-stimulation of THP-1 cells significantly increased the expression of several MMP compared to untreated cells; 4) Sulodexide treatment of both LPS- and WF-stimulated THP-1 significantly down-regulated the release of several MMPs. Our study provides evidence-based medicine during treatment of patients with VLU. WF from Infl and Gran VLU have different MMP and TIMP signatures, consistent with their clinical state. The modulation of proteolytic pathways in wound microenvironment by glycosaminoglycan sulodexide, provide insights for translating research into clinical practice during VLU therapy.

Regulation of transcription is vitally important for maintaining normal cellular homeostasis and is also the basis for cellular differentiation, morphogenesis and the adaptability of any organism. Transcription activators, which orchestrate time and locus-specific assembly of complex transcription machinery, act as key players in these processes. One way in which these activators are controlled is by the covalent attachment of the conserved protein, ubiquitin (Ub), which can serve as either a proteolytic or non-proteolytic signal. For a subset of the activators, polyubiquitination-dependent degradation of the activator controls its abundance. In these cases transcription activation can require protein synthesis as well as internal or external stimulus. In contrast, other activators have been reported to undergo mono- or oligoubiquitination that does not lead to protein degradation. The mechanisms by which monoubiquitination of transcription activators affect their activities have been poorly understood. In a recent study, we demonstrated that monoubiquitination of some transcription activators can inhibit transcription by recruiting the AAA+ ATPase Cdc48 (also known in metazoan organisms as p97 or valosin-contain protein, VCP), which then extracts the ubiquitinated activator from DNA. PMID:28357251

A new method has been described for the isolation of factor VIII. The method results in a high yield of factor VIII that is homogeneous by several different criteria. The purified protein is very stable and is not dissociated in the presence of 1 M NaCl or 0.25 M CaCl2. The highly purified protein is readily activated and inactivated by various proteolytic enzymes, such as thrombin, plasmin, and trypsin. The molecular events that lead to the activation reaction, however, have not been established.

Human tissue kallikreins (KLKs or kallikrein-related peptidases) are a subgroup of extracellular serine proteases that act on a wide variety of physiological substrates, while they display aberrant expression patterns in certain types of cancer. Differential expression patterns lead to the exploitation of these proteins as new cancer biomarkers for hormone-dependent malignancies, in particular. The prostate-specific antigen or kallikrein-related peptidase 3 (PSA/KLK3) is an established tumor marker for the diagnosis and monitoring of prostate cancer. It is well documented that specific KLK genes are co-expressed in tissues and in various pathologies suggesting their participation in complex proteolytic cascades. Here, we review the currently established knowledge on the involvement of KLK proteolytic cascades in the regulation of physiological and pathological processes in prostate tissue and in skin. It is well established that the activity of KLKs is often regulated by auto-activation and subsequent autolytic internal cleavage leading to enzymatic inactivation, as well as by inhibitory serpins or by allosteric inhibition by zinc ions. Redistribution of zinc ions and alterations in their concentration due to physiological or pathological reasons activates specific KLKs initiating the kallikrein cascade(s). Recent studies on kallikrein substrate specificity allowed for the construction of a kallikrein interaction network involved in semen liquefaction and prostate cancer, as well as in skin pathologies, such as skin desquamation, psoriasis and cancer. Furthermore, we discuss the crosstalks between known proteolytic pathways and the kallikrein cascades, with emphasis on the activation of plasmin and its implications in prostate cancer. These findings may have clinical implications for the underlying molecular mechanism and management of cancer and other disorders in which KLK activity is elevated.

A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolyticactivity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (Ea), quotient energy (Q10), Km, and Vmax were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively. PMID:26989505

Proteolytic signalling cascades control a wide range of physiological responses. In order to respond rapidly, protease activity must be maintained at a basal level: the component zymogens must be sequentially activated and actively degraded. At the same time, signalling cascades must respond precisely: high target specificity is required. The insects have a wide range of trapping- and tight-binding protease inhibitors, which can regulate the activity of individual proteases. In addition, the interactions between component proteases of a signalling cascade can be modified by serine protease homologues. The suicide-inhibition mechanism of serpin family inhibitors gives rapid turnover of both protease and inhibitor, but target specificity is inherently broad. Similarly, the TEP/macroglobulins have extremely broad target specificity, which suits them for roles as hormone transport proteins and sensors of pathogenic virulence factors. The tight-binding inhibitors, on the other hand, have a lock-and-key mechanism capable of high target specificity. In addition, proteins containing multiple tight-binding inhibitory domains may act as scaffolds for the assembly of signalling complexes. Proteolytic cascades regulated by combinations of different types of inhibitor could combine the rapidity of suicide-inhibitors with the specificity lock-and-key inhibitors. This would allow precise control of physiological responses and may turn out to be a general rule.

Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

The hexadecapeptide cerebellin is present in the brains of many vertebrate species and is derived from a larger protein, Cbln1 (cerebellin 1 precursor protein). Although cerebellin has features of a neuropeptide, Cbln1 belongs to the C1q/tumor necrosis factor superfamily of secreted proteins, suggesting that it is the biologically active molecule and the proteolytic events that generate cerebellin serve another function. Therefore, we assessed whether Cbln1 undergoes proteolytic processing and determined what consequences the cleavage events necessary to produce cerebellin have on the structure of Cbln1. Substantial degradation of Cbln1 was evident in the synaptic compartment of cerebellum and lysates of cultured cerebellar neurons and cells transfected with Cbln1 expression vectors. However, only uncleaved Cbln1 containing the cerebellin motif was released and assembled into hexameric complexes. Using yeast two hybrid and mammalian expression systems we show that the cleavages required to produce cerebellin influence the subunit stoichiometry of Cbln1 complexes. Cleavage at the N-terminus of the cerebellin sequence in Cbln1 yields trimeric complexes by separating the trimer-mediating C-terminal C1q domain from conserved N-terminal cysteine residues that mediate higher order oligomerization. Cleavage at the C-terminus of the cerebellin motif disrupts the C1q domain and abolishes subunit interactions. Functional implications of these data are discussed.

Maintaining the body’s levels of iron within precise boundaries is essential for normal physiological function. Alterations of these levels below or above the healthy limit lead to a systemic deficiency or overload in iron. The type-two transmembrane serine protease (TTSP), matriptase-2 (also known as TMPRSS6), is attracting significant amounts of interest due to its recently described role in iron homeostasis. The finding of this regulatory role for matriptase-2 was originally derived from the observation that mice deficient in this protease present with anemia due to elevated levels of hepcidin and impaired intestinal iron absorption. Further in vitro analysis has demonstrated that matriptase-2 functions to suppress bone morphogenetic protein stimulation of hepcidin transcription through cell surface proteolytic processing of the bone morphogenetic protein co-receptor hemojuvelin. Consistently, the anemic phenotype of matriptase-2 knockout mice is mirrored in humans with matripase-2 mutations. Currently, 14 patients with iron-refractory iron deficiency anemia (IRIDA) have been reported to harbor various genetic mutations that abrogate matriptase-2 proteolyticactivity. In this review, after overviewing the membrane anchored serine proteases, in particular the TTSP family, we summarize the identification and characterization of matriptase-2 and describe its functional relevance in iron metabolism. PMID:19377077

Work involving the transforming protein, p60v-src, of Rous sarcoma virus has resulted in the extensive characterization of its protein structure and associated phosphotransferase activity. However, in many investigations proteolytic fragments (principally p52v-src) of the src protein are actually studied. Here, we emphasize potential problems in the interpretation of experimental results in which the proteolytic fragmentation of p60v-src may be involved and offer several means for the complete prevention of this p60v-src degradation. Images PMID:6306278

Saponins are naturally occurring metabolites present in Yerba mate (Ilex paraguariensis), and other plant sources which have been associated with several health benefits. Mate saponins were extracted with methanol from dry leaves, partially purified and quantified. UV-HPLC analysis showed that the m...

In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing its protein stability whereas other Bcl-2 family members were not altered. In addition, the treatment with pycnogenol led to the production of reactive oxygen species and N-acetyl-l-cysteine almost blocked pycnogenol-induced reactive oxygen species generation. Taken together, these findings suggest that pycnogenol may be a potential candidate for the chemoprevention or chemotherapy of human oral cancer. PMID:26798196

In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing its protein stability whereas other Bcl-2 family members were not altered. In addition, the treatment with pycnogenol led to the production of reactive oxygen species and N-acetyl-l-cysteine almost blocked pycnogenol-induced reactive oxygen species generation. Taken together, these findings suggest that pycnogenol may be a potential candidate for the chemoprevention or chemotherapy of human oral cancer.

The bioprospection for cellulase and protease producers is a promise strategy for the discovery of potential biocatalysts for use in hydrolysis of lignocellulosic materials as well as proteic residues. These enzymes can increment and turn viable the production of second generation ethanol from different and alternative sources. In this context, the goal of this study was the investigation of cellulolytic and proteolytic abilities of bacteria isolated from the gastrointestinal tract of a hippopotamus as well as from its composting process. It is important to highlight that hippopotamus gastrointestinal samples were a non-typical sources of efficient hydrolytic bacteria with potential for application in biotechnological industries, like biofuel production. Looking for this, a total of 159 bacteria were isolated, which were submitted to qualitative and quantitative enzymatic assays. Proteolytic analyzes were conducted through the evaluation of fluorescent probes. Qualitative assays for cellulolytic abilities revealed 70 positive hits. After quantitative analyzes, 44 % of these positive hits were selected, but five (5) strains showed cellulolytic activity up to 11,8 FPU/mL. Regarding to proteolyticactivities, six (6) strains showed activity above 10 %, which overpassed results described in the literature. Molecular analyzes based on the identification of 16S rDNA, revealed that all the selected bacterial isolates were affiliated to Bacillus genus. In summary, these results strongly indicate that the isolated bacteria from a hippopotamus can be a potential source of interesting biocatalysts with cellulolytic and proteolyticactivities, with relevance for industrial applications.

Canavanine arrests a critical function in head morphogenesis and the potential for forming giant T-even phage particles termed lollipops is induced. Formation of the particles requires the addition of arginine and the restoration of normal functions. We now report on an investigation into the effects of canavanine on both the T4-induced proteolyticactivity and on the substrate proteins. Using an in vitro cleavage assay we have shown that the gene 21-dependent proteolyticactivity from canavanine-treated extracts is markedly inhibited, whereas the substrate proteins retain a high susceptibility for cleavage. The proteolyticactivity in extracts treated with canavanine followed by arginine is readily detectable, and proteins previously synthesized in the presence of canavanine can be cleaved. Protein synthesis is apparently required for the appearance of the proteolyticactivity after the canavanine-arginine treatment. Mixing experiments suggest the requirement for a component of the gene 21-dependent proteolyticactivity that is not coded for by gene 21. Images PMID:1185853

A proteolytic enzyme, ingobsin , purified from rat duodenal extracts is shown to be localised to intestinal goblet cells of both man and rat. Enzyme positive cells decrease in number from duodenum to colon. The enzyme is a 33 000 Mr protein with an isoelectric point of 5.1. The pH optimum for enzymatic activity is 7.4-8.0. Based on substrate specificity for arg-x, lys-x and to a lesser degree tyr-x, on the effect of diisopropylphosphorofluoride , Trasylol and phenylmethylsulfonylfluoride and on proteolyticactivity towards intact proteins, ingobsin is classified as a serine proteinase with endoproteolytic activity. Images Fig. 2 Fig. 4 Fig. 6 PMID:6735249

Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences. PMID:24970197

To investigate the intestinal absorption of a fibrinolytic and proteolytic lumbrokinase extracted from Eisenia andrei, we used rat everted gut sacs and an in situ closed-loop recirculation method. We extracted lumbrokinase from Eisenia andrei, and then raised polyclonal antibody against lumbrokinase. Fibrinolytic activity and proteolyticactivity in the serosal side of rat everted gut sacs incubated with lumbrokinase showed dose- and time-dependent patterns. Immunological results obtained by western blotting serosal side solution using rat everted gut sacs method showed that lumbrokinase proteins between 33.6 and 54.7 kDa are absorbed mostly by the intestinal epithelium. Furthermore, MALDI-TOF mass spectrometric analysis of plasma fractions obtained by in situ recirculation method confirmed that lumbrokinase F1 is absorbed into blood. These results support the notion that lumbrokinase can be absorbed from mucosal lumen into blood by oral administration. PMID:20473377

All morphogens of the Hedgehog (Hh) family are synthesized as dual-lipidated proteins, which results in their firm attachment to the surface of the cell in which they were produced. Thus, Hh release into the extracellular space requires accessory protein activities. We suggested previously that the proteolytic removal of N- and C-terminal lipidated peptides (shedding) could be one such activity. More recently, the secreted glycoprotein Scube2 (signal peptide, cubulin domain, epidermal-growth-factor-like protein 2) was also implicated in the release of Shh from the cell membrane. This activity strictly depended on the CUB domains of Scube2, which derive their name from the complement serine proteases and from bone morphogenetic protein-1/tolloid metalloproteinases (C1r/C1s, Uegf and Bmp1). CUB domains function as regulators of proteolyticactivity in these proteins. This suggested that sheddases and Scube2 might cooperate in Shh release. Here, we confirm that sheddases and Scube2 act cooperatively to increase the pool of soluble bioactive Shh, and that Scube2-dependent morphogen release is unequivocally linked to the proteolytic processing of lipidated Shh termini, resulting in truncated soluble Shh. Thus, Scube2 proteins act as protease enhancers in this setting, revealing newly identified Scube2 functions in Hh signaling regulation.

Luciferase is most widely used bioluminescence protein in biotechnological processes, but the enzyme is susceptible to proteolytic degradation, thereby its intracellular half-life decreased. Osmolytes are known to enhance the stability of proteins and protect them in a native folded and functional state. The effects of osmolytes, including sucrose, glycine and DMSO on the stability of luciferase were investigated. To different extents, all osmolytes protected the luciferase towards proteolytic degradation in a concentration-dependent manner. The results showed that 1.5M sucrose, 1.5M glycine and 15% DMSO are the best. The ability of these osmolytes to protect luciferase against proteolysis decreased from sucrose, glycine, and finally DMSO. Enzymatic kinetic data showed that the luciferase activity is significantly kept in the presence of sucrose and glycine compared to DMSO, particularly at high temperatures. Bioluminescence intensity, circular dichroism (CD), intrinsic and ANS fluorescence experiments showed change in secondary and tertiary luciferase structure. These results suggest that osmolytes exert an important effect on stabilization of luciferase conformation; decreasing the unfolding rate, preventing adaptation and binding of luciferase at the active site of proteases, thereby the proteolytic digestion reduced and its active conformation was kept.

Milk contains elements of numerous proteolytic systems (zymogens, active proteases, protease inhibitors and protease activators) produced in part from blood, in part by mammary epithelial cells and in part by immune cell secretion. Researchers have examined milk proteases for decades, as they can cause major defects in milk quality and cheese production. Most previous research has examined these proteases with the aim to eliminate or control their actions. However, our recent peptidomics research demonstrates that these milk proteases produce specific peptides in healthy milk and continue to function within the infant’s gastrointestinal tract. These findings suggest that milk proteases have an evolutionary function in aiding the infant’s digestion or releasing functional peptides. In other words, the mother provides the infant with not only dietary proteins but also the means to digest them. However, proteolysis in the milk is controlled by a balance of protease inhibitors and protease activators so that only a small portion of milk proteins are digested within the mammary gland. This regulation presents a question: If proteolysis is beneficial to the infant, what benefits are gained by preventing complete proteolysis through the presence of protease inhibitors? In addition to summarizing what is known about milk proteolytic systems, we explore possible evolutionary explanations for this proteolytic balance. PMID:26179272

Proteasome complexes play essential roles in maintaining cellular protein homeostasis and serve fundamental roles in cardiac function under normal and pathological conditions. A functional detriment in proteasomal activities has been recognized as a major contributor to the progression of cardiovascular diseases. Therefore, approaches to restore proteolytic function within the setting of the diseased myocardium would be of great clinical significance. In this study, we discovered that the cardiac proteasomal activity could be regulated by acetylation. Histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid and sodium valproate) enhanced the acetylation of 20S proteasome subunits in the myocardium and led to an elevation of proteolytic capacity. This regulatory paradigm was present in both healthy and acutely ischemia/reperfusion (I/R) injured murine hearts, and HDAC inhibition in vitro restored proteolytic capacities to baseline sham levels in injured hearts. This mechanism of regulation was also viable in failing human myocardium. With 20S proteasomal complexes purified from murine myocardium treated with HDAC inhibitors in vivo, we confirmed that acetylation of 20S subunits directly, at least in part, presents a molecular explanation for the improvement in function. Furthermore, using high-resolution LC-MS/MS, we unraveled the first cardiac 20S acetylome, which identified the acetylation of nine N-termini and seven internal lysine residues. Acetylation on four lysine residues and four N-termini on cardiac proteasomes were novel discoveries of this study. In addition, the acetylation of five lysine residues was inducible via HDAC inhibition, which correlated with the enhancement of 20S proteasomal activity. Taken as a whole, our investigation unveiled a novel mechanism of proteasomal function regulation in vivo and established a new strategy for the potential rescue of compromised proteolytic function in the failing heart using HDAC inhibitors.

Alzheimer’s disease (AD) is ultimately linked to the amyloid precursor protein (APP). However, current research reveals an important synaptic function of APP and APP-like proteins (APLP1 and 2). In this context various neurotrophic and neuroprotective functions have been reported for the APP proteolytic fragments sAPPα, sAPPβ and the monomeric amyloid-beta peptide (Aβ). APP is a metalloprotein and binds copper and zinc ions. Synaptic activity correlates with a release of these ions into the synaptic cleft and dysregulation of their homeostasis is linked to different neurodegenerative diseases. Metal binding to APP or its fragments affects its structure and its proteolytic cleavage and therefore its physiological function at the synapse. Here, we summarize the current data supporting this hypothesis and provide a model of how these different mechanisms might be intertwined with each other. PMID:28197076

Heart tissue becomes zinc-depleted and the capacity to mobilize labile zinc is diminished, indicating zinc dyshomeostasis during ischemia/reperfusion (I/R). Apparently, zinc pyrithione restores the basal zinc levels during I/R and prevents apoptosis by activating phosphatidyl inositol-3-kinase/Akt and targeting mitochondrial permeability transition. Receptor tyrosine kinases of the ErbB family (ErbB1 to ErbB4) are cell surface proteins that can regulate cell growth, proliferation and survival. Previous studies have shown that zinc pyrithione-induced activation of PI3kinase/Akt requires ErbB2 expression. On the other hand, while I/R decreases ErbB2 levels causing cardiomyocyte dysfunction and cell death, zinc pyrithione restores ErbB2 levels and maintains cardiomyocyte function. H9c2 cells expressed all the four ErbBs, although the expression of ErbB1 and ErbB2 were higher compared to ErbB3 and ErbB4. Hypoxia/Reoxygenation (H/R) had opposing effects on the mRNA expression of ErbB1 and ErbB2. ErbB2 mRNA levels were enhanced, but corresponding ErbB2 protein levels decreased after reoxygenation. H/R induced the degradation of ErbB2 in caspase-3dependent manner, with the formation of a 25kDa fragment. This fragment could be detected after H/R only upon treatment of the cells with a proteasomal inhibitor, ALLN, suggesting that caspase-mediated cleavage of 185kDa ErbB2 results in C-terminal cleavage and formation of 25kDa fragment, which is further degraded by proteasome. Heterodimerization and phosphorylation of ErbB2/ErbB1 which decreased upon reoxygenation, was promoted by zinc pyrithione. Zinc pyrithione effectively suppressed the caspase activation, decreased the proteolytic cleavage of ErbB2, enhanced the phosphorylation and activation of ErbB1-ErbB2 complexes and improved the cell survival after hypoxia/reoxygenation.

The objective of this experiment was to determine whether the caspase proteolytic system has a role in postmortem tenderization. Six ewes and six wethers that were non-carriers and six ewes and six wethers that were expressing the callipyge gene were used for this study. Caspase activities were de...

Members of the extended interleukin-1 (IL-1) cytokine family, such as IL-1, IL-18, IL-33, and IL-36, play a pivotal role in the initiation and amplification of immune responses. However, deregulated production and/or activation of these cytokines can lead to the development of multiple inflammatory disorders. IL-1 family members share a broadly similar domain organization and receptor signaling pathways. Another striking similarity between IL-1 family members is the requirement for proteolytic processing in order to unlock their full biological potential. Although much emphasis has been put on the role of caspase-1, another emerging theme is the involvement of neutrophil- and mast cell-derived proteases in IL-1 family cytokine processing. Elucidating the regulation of IL-1 family members by proteolytic processing is of great interest for understanding inflammation and immunity. Here, we review the identity of the proteases involved in the proteolytic processing of IL-1 family cytokines and the therapeutic implications in inflammatory disease.

Proteolytic cascades are deeply involved in critical stages of cancer progression. During the course of peptide-wise analysis of shotgun proteomic data sets representative of colon adenocarcinoma (AC) and ulcerative colitis (UC), we detected a cancer-specific proteolytic fingerprint composed of a set of numerous protein fragments cleaved C-terminally to V, I, A, T, or C residues, significantly overrepresented in AC. A peptide set linked by a common VIATC cleavage consensus was the only prominent cancer-specific proteolytic fingerprint detected. This sequence consensus indicated neutrophil elastase as a source of the fingerprint. We also found that a large fraction of affected proteins are RNA processing proteins associated with the nuclear fraction and mostly cleaved within their functionally important RNA-binding domains. Thus, we detected a new class of cancer-specific peptides that are possible markers of tumor-infiltrating neutrophil activity, which often correlates with the clinical outcome. Data are available via ProteomeXchange with identifiers: PXD005274 (Data set 1) and PXD004249 (Data set 2). Our results indicate the value of peptide-wise analysis of large global proteomic analysis data sets as opposed to protein-wise analysis, in which outlier differential peptides are usually neglected.

The encephalomyocarditis virus 3C protease has been shown to be rapidly degraded in infected cells and in vitro in rabbit reticulocyte lysate. The in vitro degradation, at least, is accomplished by a virus-independent, ATP-dependent proteolytic system. Here we identify this proteolytic system as the ubiquitin-mediated system. Incubation of the 3C protease in rabbit reticulocyte or cultured mouse cell lysate preparations, alone or in the presence of added ubiquitin or methylated ubiquitin, resulted in the generation of new higher molecular weight species. These new products were shown to be 3C protease-ubiquitin conjugates by their ability to bind antibodies against both the 3C protease and ubiquitin. Supplemental ubiquitin also stimulated the degradation of the 3C protease in these preparations. Large 3C protease-polyubiquitin conjugates were observed to accumulate in reticulocyte lysate in the presence of adenosine 5'-O-(3-thiotriphosphate), an inhibitor of the 26 S multicatalytic protease. This, combined with the fact that the proteolyticactivity could be removed from the lysate by sedimentation, implicates the multicatalytic protease in the degradation of the 3C protease-ubiquitin conjugates. It was also found that the slow rate of degradation of a model polyprotein, which resembles the stable viral 3CD diprotein produced in vivo, is likely due to the fact that the polyprotein is a poor substrate for the ubiquitin-conjugating system.

Mucositis, a common toxic side effect of chemotherapy, is characterized by an arrest of cell proliferation and a loss of gut barrier function, which may cause treatment reduction or withdrawal. Gut integrity depends on nutritional and metabolic factors, including the balance between protein synthesis and proteolysis. The effects of methotrexate (MTX; a frequently used chemotherapeutic agent) on intestinal proteolysis and gut barrier function were investigated in rats. Male Sprague-Dawley rats received 2.5 mg/kg of MTX subcutaneously during 3 days and were euthanized at Day 4 (D4) or Day 7 (D7). We observed at D4 that MTX induced mucosal damage and increased intestinal permeability (7-fold) and the mucosal concentration of interleukin (IL)-1beta and IL-6 (4- to 6-fold). In addition, villus height and glutathione content significantly decreased. Intestinal proteolysis was also affected by MTX as cathepsin D activity increased at D4, whereas chymotrypsin-like proteasome activity decreased and calpain activities remained unaffected. At D7, cathepsin D activity was restored to control levels, but proteasome activity remained reduced. This disruption of proteolysis pathways strongly contributed to mucositis and requires further study. Lysosomal proteolyticactivity may be considered the main proteolytic pathway responsible for alteration of mucosal integrity and intestinal permeability during mucositis, as cathepsin D activity was found to be correlated with mucosal atrophy and intestinal permeability. Proteasome regulation could possibly be an adaptive process for survival. Future investigation is warranted to target proteolytic pathways with protective nutritional or pharmacological therapies during mucositis.

Mutations in either of two presenilin genes can cause familial Alzheimer's disease. Presenilins have both proteolysis-dependent functions, as components of the γ-secretase complex, and proteolysis-independent functions in signalling. In this study, we investigate a conserved function of human presenilins in the development of the simple model organism Dictyostelium discoideum. We show that the block in Dictyostelium development caused by the ablation of both Dictyostelium presenilins is rescued by the expression of human presenilin 1, restoring the terminal differentiation of multiple cell types. This developmental role is independent of proteolyticactivity, because the mutation of both catalytic aspartates does not affect presenilin ability to rescue development, and the ablation of nicastrin, a γ-secretase component that is crucial for proteolyticactivity, does not block development. The role of presenilins during Dictyostelium development is therefore independent of their proteolyticactivity. However, presenilin loss in Dictyostelium results in elevated cyclic AMP (cAMP) levels and enhanced stimulation-induced calcium release, suggesting that presenilins regulate these intracellular signalling pathways. Our data suggest that presenilin proteins perform an ancient non-proteolytic role in regulating intracellular signalling and development, and that Dictyostelium is a useful model for analysing human presenilin function.

The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored cellular receptor (uPAR) promotes plasminogen activation and the efficient generation of pericellular proteolyticactivity. We demonstrate here that expression of the tetraspanin CD82/KAI1 (a tumor metastasis suppressor) leads to a profound effect on uPAR function. Pericellular plasminogen activation was reduced by approximately 50-fold in the presence of CD82, although levels of components of the plasminogen activation system were unchanged. uPAR was present on the cell surface and molecularly intact, but radioligand binding analysis with uPA and anti-uPAR antibodies revealed that it was in a previously undetected cryptic form unable to bind uPA. This was not due to direct interactions between uPAR and CD82, as they neither co-localized on the cell surface nor could be co-immunoprecipitated. However, expression of CD82 led to a redistribution of uPAR to focal adhesions, where it was shown by double immunofluorescence labeling to co-localize with the integrin alpha(5)beta(1), which was also redistributed in the presence of CD82. Co-immunoprecipitation experiments showed that, in the presence of CD82, uPAR preferentially formed stable associations with alpha(5)beta(1), but not with a variety of other integrins, including alpha(3)beta(1). These data suggest that CD82 inhibits the proteolytic function of uPAR indirectly, directing uPAR and alpha(5)beta(1) to focal adhesions and promoting their association with a resultant loss of uPA binding. This represents a novel mechanism whereby tetraspanins, integrins, and uPAR, systems involved in cell adhesion and migration, cooperate to regulate pericellular proteolyticactivity and may suggest a mechanism for the tumor-suppressive effects of CD82/KAI1.

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system. PMID:24031904

A novel mass-spectrometric technique is described that permits the identification of the C-terminal peptide of a protein. The technique involves the incorporation of 18O into all alpha-carboxy groups liberated during enzyme-catalysed partial hydrolysis of the protein, followed by mass spectrometry to identify as the C-terminal peptide the only peptide that did not incorporate any 18O. The technique has been used to identify the true C-terminal tryptic peptide of a bacterially produced gamma-interferon and to distinguish it from a peptide produced by anomalous tryptic cleavage. It was found that a closely similar sequence segment of bacterially produced alpha 2-interferon undergoes an analogous cleavage. The technique was also used to identify the C-terminus of a clipped gamma-interferon that retains full antiviral activity. PMID:6418141

The human integrin glycoprotein (GP)IIb/IIIa plays a central role in haemostasis as an inducible receptor for fibrinogen and other RGD-containing adhesive proteins at the platelet plasma membrane. Expression of the fibrinogen receptor on platelet activation involves conformational changes in the quaternary structure of GPIIb/IIIa. Little is known, however, about the nature of this conformational transition. Given that isolated GPIIb/IIIa contains a mixture of RGD-binding and non-RGD-binding heterodimers, we used limited proteolysis as a tool for investigating the structural differences between the two conformers. Comparison of their fragmentation patterns shows that, whereas in the non-RGD-binding form of GPIIb/IIIa the N-terminal half of the heavy chain of GPIIb (GPIIbH) and the central region of GPIIIa are cleaved by endoproteinase Arg-C, these domains associate tightly with one another in the RGD-binding GPIIb/IIIa and are thus protected from proteolysis. In addition, the C-terminal half of GPIIb becomes more susceptible to degradation in the non-RGD-binding GPIIb/IIIa conformer. Our interpretation, in the context of available structural and functional data, is that a major relative reorientation of the GPIIbH and GPIIIa extracellular domains takes place along the subunit interface during the conformational transition of the platelet integrin. Images Figure 1 PMID:8129707

Incretin-based peptides are effective therapeutics for treating type 2 diabetes mellitus (T2DM). Oxyntomodulin (OXM), a dual agonist of GLP-1R and GCGR, has shown superior weight loss and glucose lowering effects, compared to single GLP-1R agonists. To overcome the short half-life and rapid renal clearance of OXM, which limit its therapeutic potential, both lipid and PEG modified OXM analogs have been reported. However, these approaches often result in reduced potency or PEG-associated toxicity. Herein we report a new class of cross-linked OXM analogs that show increased plasma stability and higher potency in activating both GLP-1R and GCGR. Moreover, the extended in vivo half-life results in superior anti-hyperglycemic activity in mice compared to the wild-type OXM. PMID:26727558

TLR9 is an innate immune receptor important for recognizing DNA of host and foreign origin. A mechanism proposed to prevent excessive response to host DNA is the requirement for proteolytic cleavage of TLR9 in endosomes to generate a mature form of the receptor (TLR9(471-1032)). We previously described another cleavage event in the juxtamembrane region of the ectodomain that generated a dominant-negative form of TLR9. Thus, there are at least two independent cleavage events that regulate TLR9. In this study, we investigated whether an N-terminal fragment of TLR9 could be responsible for regulation of the mature or negative-regulatory form. We show that TLR9(471-1032), corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by coexpression of the N-terminal fragment (TLR9(1-440)), inclusion of the hinge region (TLR9(441-1032)), or overexpression of UNC93B1, the last of which is critical for trafficking and cleavage of TLR9. TLR9(1-440) coimmunoprecipitates with full-length TLR9 and TLR9(471-1032) but does not rescue the native glycosylation pattern; thus, inappropriate trafficking likely explains why TLR9(471-1032) is nonfunctional. Lastly, we show that TLR9(471-1032) is also a dominant-negative regulator of TLR9 signaling. Together, these data provide a new perspective on the complexity of TLR9 regulation by proteolytic cleavage and offer potential ways to inhibit activity through this receptor, which may dampen autoimmune inflammation.

Plant proteases are capable of performing several functions in biological systems, and their use is attractive for biotechnological process due to their interesting catalytic properties. Bromelia pinguin (aguama) is a wild abundant natural resource in several regions of Central America and the Caribbean Islands but is underutilized. Their fruits are rich in proteases with properties that are still unknown, but they represent an attractive source of enzymes for biotechnological applications. Thus, the proteolyticactivity in enzymatic crude extracts (CEs) from wild B. pinguin fruits was partially characterized. Enzymes in CEs showed high proteolyticactivity at acid (pH 2.0-4.0) and neutral alkaline (pH 7.0-9.0) conditions, indicating that different types of active proteases are present. Proteolyticactivity inhibition by the use of specific protease inhibitors indicated that aspartic, cysteine, and serine proteases are the main types of proteases present in CEs. Activity at pH 3.0 was stable in a broad range of temperatures (25-50 °C) and retained its activity in the presence of surfactants (SDS, Tween-80), reducing agents (DTT, 2-mercapoethanol), and organic solvents (methanol, ethanol, acetone, 2-propanol), which suggests that B. pinguin proteases are potential candidates for their application in brewing, detergent, and pharmaceutical industries.

Proteases are enzymes that catalyse hydrolysis of peptide bonds thereby controlling the shape, size, function, composition, turnover and degradation of other proteins. In microbes, proteases are often identified as important virulence factors and as such have been targets for novel drug design. It is emerging that some proteases possess additional non-proteolytic functions that play important roles in host epithelia adhesion, tissue invasion and in modulating immune responses. These additional "moonlighting" functions have the potential to obfuscate data interpretation and have implications for therapeutic design. Moonlighting enzymes comprise a subcategory of multifunctional proteins that possess at least two distinct biological functions on a single polypeptide chain. Presently, identifying moonlighting proteins relies heavily on serendipitous empirical data with clues arising from proteins lacking signal peptides that are localised to the cell surface. Here, we describe examples of microbial proteases with additional non-proteolytic functions, including streptococcal pyrogenic exotoxin B, PepO and C5a peptidases, mycoplasmal aminopeptidases, mycobacterial chaperones and viral papain-like proteases. We explore how these non-proteolytic functions contribute to host cell adhesion, modulate the coagulation pathway, assist in non-covalent folding of proteins, participate in cell signalling, and increase substrate repertoire. We conclude by describing how proteomics has aided in moonlighting protein discovery, focusing attention on potential moonlighters in microbial exoproteomes.

Endogenous enzymes may be involved in the biodegradation of adhesive restoration-tooth interfaces. Inhibitors of matrix metalloproteinases (MMPs) have been suggested to retard the bond-degradation process. Limited data are available on whether composite cements may also benefit from MMP inhibitors. Therefore, the aim of this study was to determine the effect of two MMP inhibitors--chlorhexidine digluconate (CHX) and galardin--on the microtensile bond strength (μTBS) of two self-adhesive composite cements to dentin. Ceramic specimens were cemented to bur-cut dentin surfaces using the self-adhesive composite cements RelyX Unicem 2 (3M ESPE) or Clearfil SA (Kuraray), or the etch-and-rinse composite cement Nexus 3 (Kerr) that served as the control. The surfaces were left untreated or were pretreated with MMP inhibitors (2% CHX or 0.2 mM galardin). The μTBS was determined 'immediately' and upon ageing (water storage for 6 months). Statistical analysis revealed a significant effect of the factors 'composite cement' and 'storage', as well as all interactions, but no effect of the MMP inhibitors. After 6 months of ageing, the μTBS decreased for all cements, except for the multistep etch-and-rinse luting composite when it was applied without MMP inhibitors. The MMP inhibitors could not prevent the decrease in μTBS upon ageing and therefore do not improve the luting durability of the composite cements tested.

We have identified proteolyticactivities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolyticactivities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolyticactivities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues.

Mechanisms involved in the reduction of blood platelet functions by various plant extract, including the grape seeds extract (rich in phenolic compounds, a mixture of about 95% oligomeric phenols; GSE) are still unclear. In the literature there are few papers describing studies on the effects of GSE on selected element of hemostasis. The aim of our study was to establish and compare the influence of GSE (at final dose of 0.625-50 µg/ml) and resveratrol (3,4',5 - trihydroxystilben), a phenolic compound synthesized in grapes and vegetables and presents in wine, which has been supposed to be beneficial for the prevention of cardiovascular events, on different steps of platelet activation. We measured the effects of GSE and resveratrol on platelet aggregation, the surface expression of P-selectin, platelet microparticle formation (PMP), and superoxide anion radicals ([Formula: see text]) production in blood platelets stimulated by TRAP and thrombin. P-selectin expression and PMP formation were measured by a flow cytometer. In gel-filtered platelets activated by thrombin or TRAP and treated with different concentrations of GSE (1.25-50 µg/ml) a significant decrease of P-selectin expression, PMP formation and platelet aggregation was observed. GSE caused also a dose-dependent reduction of [Formula: see text] produced in platelets activated by TRAP or thrombin. Our present results indicate that GSE inhibits platelet signaling pathways trigged by both proteolytic (thrombin) and non-proteolytic agonist (TRAP). In the comparative studies, GSE was found to be more effective antiplatelet factor, than the solution of pure resveratrol. Thus, the polyphenol-rich extract from grape seeds can be useful as the protecting factor against cardiovascular diseases.

Mutations in CAPN3/Capn3, which codes for skeletal muscle-specific calpain-3/p94 protease, are responsible for limb-girdle muscular dystrophy type 2A. Using "knock-in" (referred to as Capn3(CS/CS)) mice, in which the endogenous calpain-3 is replaced with a mutant calpain-3:C129S, which is a proteolytically inactive but structurally intact calpain-3, we demonstrated in our previous studies that loss of calpain-3 protease activity causes muscular dystrophy [Ojima, K. et al. (2010) J. Clin. Invest. 120, 2672-2683]. However, compared to Capn3-null (Capn3(-/-)) mice, Capn3(CS/CS) mice showed less severe dystrophic symptoms. This suggests that calpain-3 also has a non-proteolytic function. This study aimed to elucidate the non-proteolytic functions of calpain-3 through comparison of Capn3(CS/CS) mice with Capn3(-/-) mice. We found that calpain-3 is a component of the sarcoplasmic reticulum (SR), and that calpain-3 interacts with, but does not proteolyze, typical SR components such as ryanodine receptor and calsequestrin. Furthermore, Capn3(CS/CS) mice showed that the nonenzymatic role of calpain-3 is required for proper Ca(2+) efflux from the SR to cytosol during muscle contraction. These results indicate that calpain-3 functions as a nonenzymatic element for the Ca(2+) efflux machinery in the SR, rather than as a protease. Thus, defects in the nonenzymatic function of calpain-3 must also be involved in the pathogenesis of limb-girdle muscular dystrophy type 2A.

A number of novel Penicillium strains belonging to Penicillium nalgiovense, Penicillium solitum, Penicillium commune, Penicillium olsonii, and Penicillium oxalicum species, isolated from the surface of traditional Greek sausages, were evaluated for their proteolytic and lipolytic potential in a solid substrate first and next in submerged fermentations, using complex media. Extracellular proteolyticactivity was assessed at acid, neutral, and alkaline pH, while the lipolytic activity was assessed using olive oil, the short-chain triacylglycerol tributyrin, and the long-chain triolein, as substrates. The study revealed that although closely related, the tested strains produce enzymes of distinct specificities. P. nalgiovense PNA9 produced the highest alkaline proteolyticactivity (13.2 unit (U)/ml) and the highest lipolytic activity with tributyrin (92 U/ml). Comparisons with known sources show that proteases and/or lipases can be secreted effectively by some Penicillia (P. nalgiovense PNA4, PNA7, and PNA9 and P. solitum PSO1), and further investigations on their properties and characteristics would be promising.

The accumulation of β-amyloid (Aβ) peptide in the brain is one of the pathological hallmarks of Alzheimer's disease and is thought to be of primary aetiological significance. In an unbiased genetic screen, we identified puromycin-sensitive aminopeptidase (PSA) as a potent suppressor of Aβ toxicity in a Drosophila model system. We established that coexpression of Drosophila PSA (dPSA) in the flies' brains improved their lifespan, protected against locomotor deficits, and reduced brain Aβ levels by clearing the Aβ plaque-like deposits. However, confocal microscopy and subcellular fractionation of amyloid-expressing 7PA2 cells demonstrated that PSA localizes to the cytoplasm. Therefore, PSA and Aβ are unlikely to be in the same cellular compartment; moreover, when we artificially placed them in the same compartment in flies, we could not detect a direct epistatic interaction. The consequent hypothesis that PSA's suppression of Aβ toxicity is indirect was supported by the finding that Aβ is not a proteolytic substrate for PSA in vitro. Furthermore, we showed that the enzymatic activity of PSA is not required for rescuing Aβ toxicity in neuronal SH-SY5Y cells. We investigated whether the stimulation of autophagy by PSA was responsible for these protective effects. However PSA's promotion of autophagosome fusion with lysosomes required proteolyticactivity and so its effect on autophagy is not identical to its protection against Aβ toxicity. PMID:23911349

The stability and activity of the HIV cell-penetrating TAT peptide (TATp) on the surface of TATp-modified micelles and liposomes in relation to its proteolytic cleavage was investigated. TATp moieties were attached to the surface of these nanocarriers using TATp modified with a conjugate of phosphatidyl ethanolamine with a ‘short’ PEG (PEG-PE). Following pre-incubation with trypsin, elastase, or collagenase, the proteolytic stability of TATp on the surface of these modified carriers was studied by HPLC with fluorescence detection using fluorenylmethyl chloroformate (FMOC) labeling. All tested enzymes produced a dose-dependent cleavage of TATp as shown by the presence of TATp Arg-Arg fragments. Inhibition of TATp cleavage occurred when these TATp-micelles were modified by the addition of longer PEG-PE blocks, indicating an effective shielding of TATp from proteolysis by these blocks. TATp-modified carriers were also tested for their ability to accumulate in EL-4, HeLa, and B16-F10 cells. Trypsin treatment of TATp-modified liposomes and micelles resulted in decreased uptake and cell interaction, as measured by fluorescence microscopy and fluorescence-activated cell sorting techniques. Furthermore, a decrease in the cytotoxicity of TATp-modified liposomes loaded with doxorubicin (Doxil) was observed following trypsin treatment. In conclusion, steric shielding of TATp is essential to ensure its in vivo therapeutic function. PMID:21438724

This work verifies that amitraz and oxalic acid treatment affect honeybee cuticle proteolytic enzymes (CPE). Three bee groups were monitored: oxalic acid treatment, amitraz treatment, control. Electrophoresis of hydrophilic and hydrophobic CPE was performed. Protease and protease inhibitor activities (in vitro) and antifungal/antibacterial efficiencies (in vivo), were analyzed. Amitraz and oxalic acid treatment reduced hydrophobic, but did not affect hydrophilic, protein concentrations and reduced both hydrophilic and hydrophobic body surface asparagine and serine protease activities in relation to most substrates and independently of pH. The activities of natural cuticle inhibitors of acidic, neutral, and alkaline proteases were suppressed as a result of the treatments, corresponding with reduced antifungal and antibacterial activity. Electrophoretic patterns of low-, medium-, and high-molecular-weight proteases and protease inhibitors were also affected by the treatments. PMID:26466630

Infectious diseases affect human health despite advances in biomedical research and drug discovery. Among these, viruses are especially difficult to tackle due to the sudden transfer from animals to humans, high mutational rates, resistance to current treatments, and the intricacies of their molecular interactions with the host. As an example of these interactions, we describe a cell-based approach to monitor specific proteolytic events executed by either the viral-encoded protease or by host proteins on the virus. We then emphasize the significance of examining proteolysis within the subcellular compartment where cleavage occurs naturally. We show the power of stable expression, highlighting the usefulness of the cell-based multiplexed approach, which we have adapted to two independent assays previously developed to monitor (a) the activity of the HIV-1-encoded protease or (b) the cleavage of the HIV-1-encoded envelope protein by the host. Multiplexing was achieved by mixing cells each carrying a different assay or, alternatively, by engineering cells expressing two assays. Multiplexing relies on the robustness of the individual assays and their clear discrimination, further enhancing screening capabilities in an attempt to block proteolytic events required for viral infectivity and spread. PMID:27688710

Phase III clinical trials with cancer patients with the first generation of synthetic MMP inhibitors (MMPIs) failed due to inefficacy and adverse side effects. These results were unexpected, given the wealth of pre-clinical data implicating MMPs as cancer targets, but are attributable to the broad-spectrum activity of these early MMPIs and the limited knowledge of the variety of biological functions of MMPs at the time they were deployed. These experiences stimulated the development of a variety of highly specific synthetic MMPIs. However, the bottle-neck is the identification of true target-MMPs. Functional genetic approaches are being complicated by the existence of the 'protease web,' i.e., the dynamic interconnectivity of MMPs and other proteases, their inhibitors, and substrates that collectively establish homeostasis in signaling in healthy and disease-afflicted tissue. Therefore, even specific MMP inhibition can result in seemingly unpredictable induction of systemic protease web-associated modulations (spam), which can comprise metastasis-promoting molecules such as other proteases and cytokines. Such undesired information in local proteolytic networks or relayed systemically in the organism via the proteolytic internet needs to be understood and defined in order to design specific metastasis therapies employing highly specific MMPIs in combination with spam-filtering agents.

The pharmacologic utility of lengthy peptides can be hindered by loss of bioactive structure and rapid proteolysis, which limits bioavailability. For example, enfuvirtide (Fuzeon, T20, DP178), a 36-amino acid peptide that inhibits human immunodeficiency virus type 1 (HIV-1) infection by effectively targeting the viral fusion apparatus, has been relegated to a salvage treatment option mostly due to poor in vivo stability and lack of oral bioavailability. To overcome the proteolytic shortcomings of long peptides as therapeutics, we examined the biophysical, biological, and pharmacologic impact of inserting all-hydrocarbon staples into an HIV-1 fusion inhibitor. We find that peptide double-stapling confers striking protease resistance that translates into markedly improved pharmacokinetic properties, including oral absorption. We determined that the hydrocarbon staples create a proteolytic shield by combining reinforcement of overall alpha-helical structure, which slows the kinetics of proteolysis, with complete blockade of peptide cleavage at constrained sites in the immediate vicinity of the staple. Importantly, double-stapling also optimizes the antiviral activity of HIV-1 fusion peptides and the antiproteolytic feature extends to other therapeutic peptide templates, such as the diabetes drug exenatide (Byetta). Thus, hydrocarbon double-stapling may unlock the therapeutic potential of natural bioactive polypeptides by transforming them into structurally fortified agents with enhanced bioavailability.

Fibrillin-1 is synthesized as a proprotein that undergoes proteolytic processing in the unique C-terminal domain by a member of the PACE/furin family of endoproteases. This family of endoproteases is active in the trans-Golgi network (TGN), but metabolic labeling studies have been controversial as to whether profibrillin-1 is processed intracellularly or after secretion. This report provides evidence that profibrillin-1 processing is not an intracellular event. Bafilomycin A1 and incubation of dermal fibroblasts at 22°C were used to block secretion in the TGN to confirm that profibrillin-1 processing did not occur in this compartment. Profibrillin-1 immunoprecipitation studies revealed that two endoplasmic reticulum-resident molecular chaperones, BiP and GRP94, interacted with profibrillin-1. To determine the proprotein convertase responsible for processing profibrillin-1, a specific inhibitor of furin, α-1-antitrypsin, Portland variant, was both expressed in the cells and added to cells exogenously. In both cases, the inhibitor blocked the processing of profibrillin-1, providing evidence that furin is the enzyme responsible for profibrillin-1 processing. These studies delineate the secretion and proteolytic processing of profibrillin-1, and identify the proteins that interact with profibrillin-1 in the secretory pathway. PMID:14523997

The chloroform extract of Physalis minima produced a significant growth inhibition against human T-47D breast carcinoma cells as compared with other extracts with an EC(50) value of 3.8 microg/mL. An analysis of cell death mechanisms indicated that the extract elicited an apoptotic cell death. mRNA expression analysis revealed the coregulation of apoptotic genes, that is, c-myc , p53, and caspase-3. The c-myc was significantly induced by the chloroform extract at the earlier phase of treatment, followed by p53 and caspase-3. Biochemical assay and ultrastructural observation displayed typical apoptotic features in the treated cells, including DNA fragmentation, blebbing and convolution of cell membrane, clumping and margination of chromatin, and production of membrane-bound apoptotic bodies. The presence of different stages of apoptotic cell death and phosphatidylserine externalization were further reconfirmed by annexin V and propidium iodide staining. Thus, the results from this study strongly suggest that the chloroform extract of P. minima induced apoptotic cell death via p53-, caspase-3-, and c-myc-dependent pathways.

Submerged and solid-state bioprocesses have been extensively explored worldwide and employed in a number of important studies dealing with microbial cultivation for the production of enzymes. The development of these production technologies has facilitated the generation of new enzyme-based products with applications in pharmaceuticals, food, bioactive peptides, and basic research studies, among others. The applicability of microorganisms in biotechnology is potentiated because of their various advantages, including large-scale production, short time of cultivation, and ease of handling. Currently, several studies are being conducted to search for new microbial peptidases with peculiar biochemical properties for industrial applications. Bioprospecting, being an important prerequisite for research and biotechnological development, is based on exploring the microbial diversity for enzyme production. Limited information is available on the production of specific proteolytic enzymes from bacterial and fungal species, especially on the subgroups threonine and glutamic peptidases, and the seventh catalytic type, nonhydrolytic asparagine peptide lyase. This gap in information motivated the present study about these unique biocatalysts. In this study, the biochemical and biotechnological aspects of the seven catalytic types of proteolytic enzymes, namely aspartyl, cysteine, serine, metallo, glutamic, and threonine peptidase, and asparagine peptide lyase, are summarized, with an emphasis on new studies, production, catalysis, and application of these enzymes.

A crude extract with high proteolyticactivity (78.1 EU/mL), prepared from ripe fruit of Bromelia antiacantha was used to hydrolyze and remove soft tissues from the epigyne of Apopyllus iheringi. This enzymatic extract presented four actives isoforms which have a broad substrate specificity action. Enzyme action on samples was optimized after evaluation under different conditions of pH, enzyme-substrate ratio and time (parameters selected based on previous studies) of treatment (pH 4.0, 6.0 and 8.0 at 42°C with different amount of enzyme). Scanning electron microscopy was used to evaluate conditions resulting in complete digestion of epigyne soft tissues. Optimal conditions for soft tissue removal were 15.6 total enzyme units, pH 6.0 for 18 h at 42°C.

We examined the functioning of the antioxidative defense system in Arabidopsis thaliana under sulphur (S) deficiency with an emphasis on the role of mitochondria. In tissue extracts and in isolated mitochondria from S-deficient plants, the concentration of non-protein thiols declined but protein thiols did not change. Superoxide anion and hydrogen peroxide were accumulated in leaf blades and the generation of superoxide anion by isolated mitochondria was higher. Lower abundance of reduced (GSH) plus oxidized (GSSG) glutathione in the leaf and root tissues, and leaf mitochondria from S-deficient plants was accompanied by a decrease in the level of GSH and the changes in the GSH/GSSG ratios. In the chloroplasts, the total level of glutathione decreased. Lower levels of reduced (AsA) and oxidized (DHA) ascorbate were reflected in much higher ratios of AsA/DHA. Sulphur deficiency led to an increase in the activity of cytosolic, mitochondrial and chloroplastic antioxidative enzymes, peroxidases, catalases and superoxide dismutases. The protein carbonyl level was higher in the leaves of S-deficient plants and in the chloroplasts, while in the roots, leaf and root mitochondria it remained unchanged. Protease activity in leaf extracts of S-deficient plants was higher, but in root extracts it did not differ. The proteolytic system reflected subcellular specificity. In leaf and root mitochondria the protease activity was higher, whereas in the chloroplasts it did not change. We propose that the preferential incorporation of S to protein thiols and activation of antioxidative and proteolytic systems are likely important for the survival of S-deficient plants and that the mitochondria maintain redox homeostasis.

Chemerin is a potent chemotactic factor that was identified recently as the ligand of ChemR23, a G protein-coupled receptor expressed by mononuclear phagocytes, dendritic cells (DCs), and NK cells. Chemerin is synthesized as a secreted precursor, prochemerin, which is poorly active on ChemR23. However, prochemerin can be converted rapidly into a full ChemR23 agonist by proteolytic removal of a carboxy-terminal peptide. This maturation step is mediated by the neutrophil-derived serine proteases elastase and cathepsin G. In the present work, we have investigated proteolytic events that negatively control chemerin activity. We demonstrate here that neutrophil-derived proteinase 3 (PR3) and mast cell (MC) chymase are involved in the generation of specific chemerin variants, which are inactive, as they do not induce calcium release or DC chemotaxis. Mass spectrometry analysis showed that PR3 specifically converts prochemerin into a chemerin form, lacking the last eight carboxy-terminal amino acids, and is inactive on ChemR23. Whereas PR3 had no effect on bioactive chemerin, MC chymase was shown to abolish chemerin activity by the removal of additional amino acids from its C-terminus. This effect was shown to be specific to bioactive chemerin (chemerin-157 and to a lesser extent, chemerin-156), as MC chymase does not use prochemerin as a substrate. These mechanisms, leading to the production of inactive variants of chemerin, starting from the precursor or the active variants, highlight the complex interplay of proteases regulating the bioactivity of this novel mediator during early innate immune responses.

Phytosulfokines (PSKs) are secreted, sulfated peptide hormones derived from larger prepropeptide precursors. Proteolytic processing of one of the precursors, AtPSK4, was demonstrated by cleavage of a preproAtPSK4-myc transgene product to AtPSK4-myc. Cleavage of proAtPSK4 was induced by placing root explants in tissue culture. The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin-like serine protease, encoded by one of 56 subtilase genes in Arabidopsis. The gene encoding AtSBT1.1 was up-regulated following the transfer of root explants to tissue culture, suggesting that activation of the proteolytic machinery that cleaves proAtPSK4 is dependent on AtSBT1.1 expression. We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity-purified AtSBT1.1. An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor. Thus, this peptide growth factor, which promotes callus formation in culture, is proteolytically cleaved from its precursor by a specific plant subtilase encoded by a gene that is up-regulated during the process of transferring root explants to tissue culture.

By mediating depolarization-induced Ca2+ influx high voltage-activated (HVA) Ca2+ channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (α1) and three auxiliary (α2δ, β and γ) subunits. The α2δ subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, α2δ must undergo important post-translational modifications including a proteolytic cleavage that separates the extracellular α2 from its transmembrane δ domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature α2δ almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of α2δ was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide reducing agent. Interestingly, these mutations significantly decreased whole-cell patch clamp currents without affecting the voltage-dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane. PMID:17408426

The proteolytic enzymes produced by Erwinia carotovora subsp. carotovora (Ecc-strain SR 394) grown on various media were examined by isoelectrofocusing in polyacrylamide gels over a pH range of 3-10. In addition to the main protease present in culture filtrates, low concentrations of several other proteases were present in extracts from potato tubers infected by Ecc. Proteases from all these sources were similar and had the following properties: pH optimum near 8.0, calcium dependent, insensitive to serine proteinase and SH-proteinase inhibitors, inhibited by EDTA, and highly thermostable. These enzymes degraded gelatin, soluble collagen and Hide Powder Azure, and showed weak activity on casein, but did not degrade insoluble collagen or elastin.

Reovirus adheres specifically to apical membranes of mouse intestinal M cells and exploits M-cell transepithelial transport activity to enter Peyer's patch mucosa, where replication occurs. Proteolytic conversion of native reovirus to intermediate subviral particles (ISVPs) occurs in the intestine, but it is not known whether conversion is essential for interaction of virus with M cells. We tested the capacity of native virions, ISVPs, and cores (that lack outer capsid proteins) to bind to intestinal epithelial cells in vivo and found that only ISVPs adhered to M cells. Thus, intraluminal conversion of native reovirus to ISVPs is a prerequisite for M-cell adherence, and outer capsid proteins unique to ISVPs (either sigma 1 or products of mu 1) mediate interaction of virus with M-cell apical membranes. Images PMID:7525989

Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3 clipping was performed by serine and aspartyl proteases in HeLa cells. These results suggest that histone H3 clipping operates as part of post-translational modification system in CC.

Wound dressings have experienced continuous and significant changes over the years based on the knowledge of the biochemical events associated with chronic wounds. The development goes from natural materials used to just cover and conceal the wound to interactive materials that can facilitate the healing process, addressing specific issues in non-healing wounds. These new types of dressings often relate with the proteolytic wound environment and the bacteria load to enhance the healing. Recently, the wound dressing research is focusing on the replacement of synthetic polymers by natural protein materials to delivery bioactive agents to the wounds. This article provides an overview on the novel protein-based wound dressings such as silk fibroin keratin and elastin. The improved properties of these dressings, like the release of antibiotics and growth factors, are discussed. The different types of wounds and the effective parameters of healing process will be reviewed.

The notorious degradation susceptibility of peptides is a major obstacle to their use as medicinal drugs. Assays with which the stability of peptides in complex proteolytic environments can be determined are thus indispensable for peptide drug development. Herein, we describe a new peptide proteolysis assay that meets that demand. It unites the high-throughput capacity of heterogeneous with the well-defined kinetic characteristics of homogeneous assay formats and operates on the cleavage-caused loss of a detection handle. We have confirmed the assay's accuracy with well-defined model interactions and proved its high versatility and robustness with a representative application where we determined the half-lives of 375 different peptides in a crude intestinal protease preparation. With this reliable, reproducible, and efficient assay the enzyme kinetics of any peptide-protease interaction is accessible even for complex protease solutions.

Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3 clipping was performed by serine and aspartyl proteases in HeLa cells. These results suggest that histone H3 clipping operates as part of post-translational modification system in CC. PMID:27698925

The stability of cold-water fish gelatin (FG), both in solution and in the gel phase, has been studied as function of both temperature and exposure towards novel proteases of marine origin. A 1% (w/v) FG solution was readily degraded by such proteases above 20 degrees C, which was expected since FG at this temperature is a random coil molecule lacking the protective triple helical structure found in collagen. The dynamic storage modulus for a 10% (w/v) FG gel increased monotonically at 4 degrees C. Ramping the temperature to 6, 8 or 10 degrees C led to a drastic reduction in G', but an apparent partial recovery of the network (increasing G') was observed with time at all temperatures. In the presence of proteases, a lower storage modulus was observed. At constant 4 degrees C, an apparent maximum value was reached after curing for 2h followed by a decrease in G' indicating protease activity. Ramping of temperature in the presence of proteases led to an even more drastic reduction in G' and no recovery of structure was observed with time. In this case, the overall rheological behaviour is a complex function of both thermal influence as well as proteolyticactivity. In an endeavour to quantify the effect of the presence of proteolytic enzymes on the gelatin network, rheological investigation were undertaken where the dynamic storage moduli were recorded on different 10% (w/v) FG samples that had been acid hydrolysed to yield different average molecular weights. A significant reduction in storage modulus for average molecular weights below 50 kDa was found. This critical molecular weight most probably reflects the on-set of a regime where shorter chain lengths prevent percolation due to an increase in the loose end and sol fraction as well as a reduction in the average length of the pyrrolidine-rich regions reducing the number of possible junction zones.

Early accurate diagnosis and personalized treatment are essential in order to treat complex or fatal diseases such as cancer and autoimmune, cardiovascular and neurodegenerative diseases. To realize this vision, new diagnostic and prognostic biomarkers are urgently required. MS-based proteomics is the most promising approach for protein biomarker identification, but suffers in clinical translation of biomarker candidates that show only quantitative differences from normal tissue. Indeed, success in translating proteomic data to biomarkers in the clinic has been disappointing. Here, we propose that protein termini provide a new opportunity for biomarker discovery due to qualitative differences in intact and new protein termini between diseased and normal tissues. Altered proteolysis occurs in most pathologies. Disease- and process-specific protein modifications, including proteolytic processing and subsequent modification of the terminal amino acids, frequently lead to altered protein activity that plays key roles in the disease process. Thus, mapping of ensembles of characteristic protein termini provides a proteolytic signature of high information content that shows both quantitative and most importantly qualitative differences in different diseases and stage of disease. These unique protein biomarkers have the added benefit of being mechanistically informative by revealing the activity state of the bioactive protein. Moreover, proteome-wide isolation of protein termini leads to generalized sample simplification, thereby enabling up to three orders of magnitude lower LODs compared to traditional shotgun proteomic approaches. We introduce the potential of protein termini for biomarker discovery, briefly review methods enabling large-scale studies of protein termini, and discuss how these may be integrated into a termini-oriented biomarker discovery pipeline from discovery to clinical application.

It has been documented that caspase-8, a central player in apoptosis, is also crucial for TCR-mediated NF-κB activation. However, whether other caspases are also involved this process is unknown. In this report, we showed that in addition to caspase-8, caspase-9 is required for TCR-mediated NF-κB activation. Caspase-9 induces activation of PKC-θ, phosphorylation of Bcl10 and NF-κB activation in a caspase-3-dependent manner, but it appears that Bcl10 phosphorylation is uncoupled from NF-κB activation. Furthermore, caspase-8 lies upstream of caspase-9 during T cell activation. Therefore, TCR ligation elicits a caspase cascade involving caspase-8, caspase-9 and caspase-3 which initiates PKC-θ-dependent pathway leading to NF-κB activation and PKC-θ-independent Bcl10 phosphorylation which limits NF-kB activity. PMID:24924627

Cancer cells manoeuvre through extracellular matrices (ECMs) using different invasion modes, including single cell and collective cell invasion. These modes rely on MMP-driven ECM proteolysis to make space for cells to move. How cancer-associated alterations in ECM influence the mode of invasion remains unclear. Further, the sensitivity of the two invasion modes to MMP dynamics remains unexplored. In this paper, we address these open questions using a multiscale hybrid computational model combining ECM density-dependent MMP secretion, MMP diffusion, ECM degradation by MMP and active cell motility. Our results demonstrate that in randomly aligned matrices, collective cell invasion is more efficient than single cell invasion. Although increase in MMP secretion rate enhances invasiveness independent of cell-cell adhesion, sustenance of collective invasion in dense matrices requires high MMP secretion rates. However, matrix alignment can sustain both single cell and collective cell invasion even without ECM proteolysis. Similar to our in-silico observations, increase in ECM density and MMP inhibition reduced migration of MCF-7 cells embedded in sandwich gels. Together, our results indicate that apart from cell intrinsic factors (i.e., high cell-cell adhesion and MMP secretion rates), ECM density and organization represent two important extrinsic parameters that govern collective cell invasion and invasion plasticity.

The amyloid precursor protein (APP) has occupied a central position in Alzheimer’s disease (AD) pathophysiology, in large part due to the seminal role of amyloid-β peptide (Aβ), a proteolytic fragment derived from APP. Although the contribution of Aβ to AD pathogenesis is accepted by many in the research community, recent studies have unveiled a more complicated picture of APP’s involvement in neurodegeneration in that other APP-derived fragments have been shown to exert pathological influences on neuronal function. However, not all APP-derived peptides are neurotoxic, and some even harbor neuroprotective effects. In this review, we will explore this complex picture by first discussing the pleiotropic effects of the major APP-derived peptides cleaved by multiple proteases, including soluble APP peptides (sAPPα, sAPPβ), various C- and N-terminal fragments, p3, and APP intracellular domain fragments. In addition, we will highlight two interesting sequences within APP that likely contribute to this duality in APP function. First, it has been found that caspase-mediated cleavage of APP in the cytosolic region may release a cytotoxic peptide, C31, which plays a role in synapse loss and neuronal death. Second, recent studies have implicated the –YENPTY– motif in the cytoplasmic region as a domain that modulates several APP activities through phosphorylation and dephosphorylation of the first tyrosine residue. Thus, this review summarizes the current understanding of various APP proteolytic products and the interplay among them to gain deeper insights into the possible mechanisms underlying neurodegeneration and AD pathophysiology. PMID:25287911

Myofibrillar protein turnover is a key component of muscle growth and degeneration, requiring proteolytic enzymes to degrade the skeletal muscle proteins. The objective of this study was to investigate the role of the calpain proteolytic system in muscle growth development using μ-calpain knockout (KO) mice in comparison with control wild-type (WT) mice, and evaluate the subsequent effects of silencing this gene on other proteolytic systems. No differences in muscle development between genotypes were observed during the early stages of growth due to the up regulation of other proteolytic systems. The KO mice showed significantly greater m-calpain protein abundance (P < 0.01) and activity (P < 0.001), and greater caspase 3/7 activity (P < 0.05). At 30 wk of age, KO mice showed increased protein:DNA (P < 0.05) and RNA:DNA ratios (P < 0.01), greater protein content (P < 0.01) at the expense of lipid deposition (P < 0.05), and an increase in size and number of fast-twitch glycolytic muscle fibers (P < 0.05), suggesting that KO mice exhibit an increased capacity to accumulate and maintain protein in their skeletal muscle. Also, expression of proteins associated with muscle regeneration (neural cell adhesion molecule and myoD) were both reduced in the mature KO mice (P < 0.05 and P < 0.01, respectively), indicating less muscle regeneration and, therefore, less muscle damage. These findings indicate the concerted action of proteolytic systems to ensure muscle protein homeostasis in vivo. Furthermore, these data contribute to the existing evidence of the importance of the calpain system's involvement in muscle growth, development, and atrophy. Collectively, these data suggest that there are opportunities to target the calpain system to promote the growth and/or restoration of skeletal muscle mass.

Tannerella forsythia is a gram-negative bacterium strongly associated with the development and/or progression of periodontal disease. Here, we have shown that a newly characterized matrix metalloprotease-like enzyme, referred to as karilysin, efficiently cleaved the antimicrobial peptide LL-37, significantly reducing its bactericidal activity. This may contribute to the resistance of T. forsythia to the antibacterial activity of LL-37, since their vitality was found not to be affected by LL-37 at concentrations up to 2.2 muM. Furthermore, proteolysis of LL-37 by karilysin not only abolished its ability to bind lipopolysaccharide (LPS) to quench endotoxin-induced proinflammatory activity, but LL-37 cleavage also caused the release of active endotoxin from the LPS/LL-37 complex. Proteolytic inactivation of LL-37 bactericidal activity by karilysin may protect LL-37-sensitive species in the subgingival plaque and maintain the local inflammatory reaction driven by LPS from gram-negative bacteria. Consequently, the karilysin protease may directly contribute to periodontal tissue damage and the development and/or progression of chronic periodontitis.

Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolyticactivities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolyticactivity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolyticactivity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolyticactivities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus

Intracellular localization has been reported for over three-quarters of receptor tyrosine kinase (RTK) families in response to environmental stimuli. Internalized RTK may bind to non-canonical substrates and affect various cellular processes. Many of the intracellular RTKs exist as fragmented forms that are generated by γ-secretase cleavage of the full-length receptor, shedding, alternative splicing, or alternative translation initiation. Soluble RTK fragments are stabilized and intracellularly transported into subcellular compartments, such as the nucleus, by binding to chaperone or transcription factors, while membrane-bound RTKs (full-length or truncated) are transported from the plasma membrane to the ER through the well-established Rab- or clathrin adaptor protein-coated vesicle retrograde trafficking pathways. Subsequent nuclear transport of membrane-bound RTK may occur via two pathways, INFS or INTERNET, with the former characterized by release of receptors from the ER into the cytosol and the latter characterized by release of membrane-bound receptor from the ER into the nucleoplasm through the inner nuclear membrane. Although most non-canonical intracellular RTK signaling is related to transcriptional regulation, there may be other functions that have yet to be discovered. In this review, we summarize the proteolytic processing, intracellular trafficking and nuclear functions of RTKs, and discuss how they promote cancer progression, and their clinical implications.

Proteolytic stability in gastrointestinal tract and blood plasma is the major obstacle for oral peptide drug development. Inhibitor cystine knots (ICKs) are linear cystine knot peptides which have multifunctional properties and could become promising drug scaffolds. ProTx-I, ProTx-II, GTx1-15, and GsMTx-4 were spider-derived ICKs and incubated with pepsin, trypsin, chymotrypsin, and elastase in physiological conditions to find that all tested peptides were resistant to pepsin, and ProTx-II, GsMTx-4, and GTx1-15 showed resistance to all tested proteases. Also, no ProTx-II degradation was observed in rat blood plasma for 24 hours in vitro and ProTx-II concentration in circulation decreased to half in 40 min, indicating absolute stability in plasma and fast clearance from the system. So far, linear peptides are generally thought to be unsuitable in vivo, but all tested ICKs were not degraded by pepsin and stomach could be selected for the alternative site of drug absorption for fast onset of the drug action. Since spider ICKs are selective inhibitors of various ion channels which are related to the pathology of many diseases, engineered ICKs will make a novel class of peptide medicines which can treat variety of bothering symptoms. PMID:26843868

Internal chemical oscillators (chemical clocks) direct the behavior of numerous biological systems, and maintenance of a given period and phase among many such oscillators may be important for their proper function. However, both environmental variability and fundamental molecular noise can cause biochemical oscillators to lose coherence. One solution to maintaining coherence is entrainment, where an external signal provides a cue that resets the phase of the oscillators. In this work, we study the entrainment of gene networks by a queueing interaction established by competition between proteins for a common proteolytic pathway. Principles of queueing entrainment are investigated for an established synthetic oscillator in Escherichia coli. We first explore this theoretically using a standard chemical reaction network model and a map-based model, both of which suggest that queueing entrainment can be achieved through pulsatile production of an additional protein competing for a common degradation pathway with the oscillator proteins. We then use a combination of microfluidics and fluorescence microscopy to verify that pulse trains modulating the production rate of a fluorescent protein targeted to the same protease (ClpXP) as the synthetic oscillator can entrain the oscillator.

Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1. PMID:27481162

Tannerella forsythia is a periodontal pathogen expressing six secretory proteolytic enzymes with a unique multidomain structure referred to as KLIKK proteases. Two of these proteases, karilysin and mirolysin, were previously shown to protect the bacterium against complement-mediated bactericidal activity. The latter metalloprotease, however, was not characterized at the protein level. Therefore, we purified recombinant mirolysin and subjected it to detailed biochemical characterization. Mirolysin was obtained as a 66 kDa zymogen, which autoproteolytically processed itself into a 31 kDa active form via truncations at both the N- and C-termini. Further autodegradation was prevented by calcium. Substrate specificity was determined by the S1' subsite of the substrate-binding pocket, which shows strong preference for Arg and Lys at the carbonyl side of a scissile peptide bond (P1' residue). The protease cleaved an array of host proteins, including human fibronectin, fibrinogen, complement proteins C3, C4, and C5, and the antimicrobial peptide, LL-37. Degradation of LL-37 abolished not only the bactericidal activity of the peptide, but also its ability to bind lipopolysaccharide (LPS), thus quenching the endotoxin proinflammatory activity. Taken together, these results indicate that, through cleavage of LL-37 and complement proteins, mirolysin might be involved in evasion of the host immune response.

The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5-7, but the aspartic proteases were inactive across a pH range of 3-10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi.

ᅟ: Matrix metalloproteinases can modulate the inflammatory response through processing of cyto- and chemokines. Among them, MMP-14 is a non-dispensable collagenase responsible for the activation of other enzymes, triggering a proteolytic cascade. To identify the role of MMP-14 during the pro-inflammatory response, wildtype and Mmp14 (-/-) mice were challenged with lipopolysaccharide. MMP-14 levels decreased after endotoxemia. Mutant animals showed 100% mortality, compared to 50% in wildtype mice. The increased mortality was related to a more severe lung injury, an impaired lung MMP-2 activation, and increased levels of the alarmin S100A9. There were no differences in the expression of other mediators including Il6, Cxcl2, Tgfb, Il10, or S100a8. A similar result was observed in lung explants of both genotypes cultured in presence of lipopolysaccharide. In this ex vivo model, exogenous activated MMP-2 ameliorated the observed increase in alarmins. Samples from septic patients showed a decrease in serum MMP-14 and activated MMP-2 compared to non-septic critically ill patients. These results demonstrate that the MMP-14-MMP-2 axis is downregulated during sepsis, leading to a proinflammatory response involving S100A9 and a more severe lung injury. This anti-inflammatory role of MMP-14 could have a therapeutic value in sepsis.

Our previous studies showed that biomodification of demineralized dentin collagen with proanthocyanidin (PA) for a clinically practical duration improves the mechanical properties of the dentin matrix and the immediate resin-dentin bond strength. The present study sought to evaluate the ability of PA biomodification to reduce collagenase-induced biodegradation of demineralized dentin matrix and dentin/adhesive interfaces in a clinically relevant manner. The effects of collagenolytic and gelatinolytic activity on PA-biomodified demineralized dentin matrix were analysed by hydroxyproline assay and gelatin zymography. Then, resin-/dentin-bonded specimens were prepared and challenged with bacterial collagenases. Dentin treated with 2% chlorhexidine and untreated dentin were used as a positive and negative control, respectively. Collagen biodegradation, the microtensile bond strengths of bonded specimens and the micromorphologies of the fractured interfaces were assessed. The results revealed that both collagenolytic and gelatinolytic activity on demineralized dentin were notably inhibited in the PA-biomodified groups, irrespective of PA concentration and biomodification duration. When challenged with exogenous collagenases, PA-biomodified bonded specimens exhibited significantly less biodegradation and maintained higher bond strengths than the untreated control. These results suggest that PA biomodification was effective at inhibiting proteolyticactivity on demineralized dentin matrix and at stabilizing the adhesive/dentin interface against enzymatic degradation, is a new concept that has the potential to improve bonding durability.

Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

Our previous studies showed that biomodification of demineralized dentin collagen with proanthocyanidin (PA) for a clinically practical duration improves the mechanical properties of the dentin matrix and the immediate resin–dentin bond strength. The present study sought to evaluate the ability of PA biomodification to reduce collagenase-induced biodegradation of demineralized dentin matrix and dentin/adhesive interfaces in a clinically relevant manner. The effects of collagenolytic and gelatinolytic activity on PA-biomodified demineralized dentin matrix were analysed by hydroxyproline assay and gelatin zymography. Then, resin-/dentin-bonded specimens were prepared and challenged with bacterial collagenases. Dentin treated with 2% chlorhexidine and untreated dentin were used as a positive and negative control, respectively. Collagen biodegradation, the microtensile bond strengths of bonded specimens and the micromorphologies of the fractured interfaces were assessed. The results revealed that both collagenolytic and gelatinolytic activity on demineralized dentin were notably inhibited in the PA-biomodified groups, irrespective of PA concentration and biomodification duration. When challenged with exogenous collagenases, PA-biomodified bonded specimens exhibited significantly less biodegradation and maintained higher bond strengths than the untreated control. These results suggest that PA biomodification was effective at inhibiting proteolyticactivity on demineralized dentin matrix and at stabilizing the adhesive/dentin interface against enzymatic degradation, is a new concept that has the potential to improve bonding durability. PMID:24810807

Neutrophils are considered the central cells of acute inflammation. Flavonoids have been suggested as therapeutic agents to avoid damages induced by inflammatory processes. It is well known the reactivity of flavonoids with hypochlorous acid produced by neutrophils, to form stable mono and dichlorinated products. In this study, we synthesized novel chlorinated flavonoids and investigated their effect in neutrophils' oxidative burst and in its lifespan, in comparison with the parent non-chlorinated flavonoids. The obtained results demonstrate that chlorinated flavonoids were more efficient than their parent compounds in modulating neutrophils' oxidative burst in phorbol myristate acetate-activated neutrophils. Some of the tested flavonoids drive neutrophil apoptosis in a caspase3-dependent fashion. The present data showed that 8-chloro-3',4',5,7-tetrahydroxyflavone (4a) constitute an alternative anti-inflammatory therapy, due to the proven ability to suppress mechanisms engaged at the onset and progression of inflammation.

Many mammalian species that interact with venomous snakes show resistances to venoms. The family Sciuridae has several North American members that harass venomous snakes and show proteolytic resistances in their sera. We examined sera collected from an African ground squirrel (Xerus inauris) against two sympatric venomous snakes (Bitis arietans and Naja annulifera) and found no support for proteolytic resistance. Our results add to our understanding of the risks in predator defense within the family Sciuridae.

Thermally-induced gels were made from white croaker (Pennahia argentata) meat in the presence of its organ extracts by pre-heating at 40 and 65°C for 20 min and subsequent heating at 85°C for 20 min. The breaking strength of the gels decreased with increasing concentrations of the intestinal extracts accompanying decomposition of myosin heavy chains. However, no significant changes in the gel strength occurred when the kidney extract was added. The proteolyticactivity in the intestinal extracts examined in the meat homogenate had a maximum at 60°C and pH 8.90. These results suggest that the intestinal rather than kidney proteolyticactivities are responsible for gel softening known as a modori phenomenon. Thus, the removal of intestinal tracts is essential to maintain a high quality of surimi-based products.

Botulinum neurotoxins (BoNTs) are the most potent toxins known. However, the paralytic effect caused by BoNT serotypes A and B is taken advantage of to treat different forms of dystonia and in cosmetic procedures. Due to the increasing areas of application, the demand for BoNTs A and B is rising steadily. Because of the high toxicity, it is mandatory to precisely determine the potency of every produced BoNT batch, which is usually accomplished by performing toxicity testing (LD50 test) in mice. Here we describe an alternative in vitro assay for the potency determination of the BoNT serotype B. In this assay, the toxin is first bound to its specific receptor molecules. After the proteolytic subunit of the toxin has been released and activated by chemical reduction, it is exposed to synaptobrevin, its substrate protein. Finally the proteolytic cleavage is quantified by an antibody-mediated detection of the neoepitope, reaching a detection limit below 0.1mouseLD50/ml. Thus, the assay, named BoNT/B binding and cleavage assay (BoNT/B BINACLE), takes into account the binding as well as the protease function of the toxin, thereby measuring its biological activity.

The conversion of cellular prion protein (PrP(C)) into its pathological isoform (PrP(Sc)) conveys an increase in hydrophobicity and induces a partial resistance to proteinase K (PK). Interestingly, co-incubation with high copper ion concentrations also modifies the solubility of PrP(c) and induces a partial PK resistance which was reminiscent of PrP(Sc). However, concerns were raised whether this effect was not due to a copper-induced inhibition of the PK itself. We have therefore analyzed the kinetics of the formation of PK-resistant PrP(C) and excluded possible interference effects by removing unbound copper ions prior to the addition of PK by methanol precipitation or immobilization of PrP(C) followed by washing steps. We found that preincubation of PrPc with copper ions at concentrations as low as 50 microM indeed rendered these proteins completely PK resistant, while control substrates were proteolyzed. No other divalent cations induced a similar effect. However, in addition to this specific stabilizing effect on PrP(C), higher copper ion concentrations in solution (>200 microM) directly blocked the enzymatic activity of PK, possibly by replacing the Ca2+ ions in the active center of the enzyme. Therefore, as a result of this inhibition the proteolytic degradation of PrP(C) as well as PrP(Sc) molecules was suppressed.

Limited papain hydrolysis of immunoglobulin M (IgM) produces a subunit-like proteolytic fragment designated IgMp (Inman & Hazen, 1968). In the presence of mercaptans, IgMp partially dissociated into Fcμ-like and Fabμ fragments. Treatment of residual IgM (that remaining after a papain digestion) with 2mm-mercaptoethylamine resulted in fragmentation of the same type that occurs in a routine limited digestion of IgM with papain, although exogenous enzyme was not added to the mixture. When IgM was hydrolysed with 14C-labelled papain, a small quantity of the enzyme was found to be associated with the residual IgM and IgMp fractions. IgM and IgM 7S subunit (IgMs) that had been exposed to papain in the absence of activating mercaptan and separated from the enzyme by gel filtration also fragmented when subsequently treated with 2mm-mercaptoethylamine. The fragments resembled those produced during a typical limited papain digestion of IgM. It was concluded that mercaptoethylamine induced fragmentation of IgMp by activating adsorbed papain. ImagesFig. 1.PLATE 1 PMID:5076228

A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolyticactivity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism. PMID:22007293

During fermentation of sour dough the flour proteins are degraded. The proteolysis depends not only on lactic acid bacteria (Lactobacillus plantarum, L. brevis ssp. lindneri, L. fructivorans) but also on the conditions of fermentation of the sour dough. An increase of temperatures between 25 degrees C and 35 degrees C causes an increase in the amino acid content. The water content of the dough (T.A. 150/T.A. 210) influences the proteolyticactivity of the bacteria to a lesser degree.

Enteric-coated proteolytic enzyme preparations like Wobenzym and Phlogenzym are widely used for the so-called 'systemic enzyme therapy' both in humans and animals. Numerous publications reveal that oral proteolytic enzymes are able to stimulate directly the activity of immune competent cells as well as to increase efficiency of some of their products. But origins of the immunostimulatory effects of oral proteolytic enzymes are still unclear. The hypothesis described here suggests that it may be proteolysis of intestinal microorganisms that makes the immune competent cells to work in the immunostimulatory manner. The hypothesis was largely formed by several scientific observations: First, microbial lysis products (lipopolysaccharides, muropeptides and other peptidoglycan fragments, beta-glucans, etc.) are well known for their immunostimulatory action. Second, a normal human being hosts a mass of intestinal microorganisms equivalent to about 1 kg. The biomass (mainly due to naturally occurring autolysis) continuously supplies the host's organism with immunostimulatory microbial cell components. Third, the immunostimulatory effects resulting from the oral application of exogenously acting antimicrobial (lytic) enzyme preparations, such as lysozyme and lysosubtilin, are likely to be a result of the action of microbial lysis products. Fourth, cell walls of most microorganisms contain a considerable amount of proteins/peptides, a possible target for exogenous proteolytic enzymes. In fact, several authors have already shown that a number of proteases possess an ability to lyse the microbial cells in vitro. Fifth, the pretreatment of microbial cells (at least of some species) in vitro with proteolytic enzymes makes them more sensitive to the lytic action of lysozyme and, otherwise, pretreatment with lysozyme makes them more susceptible to proteolytic degradation. Sixth, exogenous proteases, when in the intestines, may participate in final steps of food-protein digestion

In this study, a risk assessment of proteolytic Clostridium botulinum in canned foie gras was performed, the number of illnesses per year in France due to C. botulinum in foie gras was estimated. Data on initial level in raw materials were collected at manufacturers and analysed using a Negative Binomial distribution. The effect of the usual foie gras heat treatment (equivalent time at 121 °C: F0=0.5 min) was considered at two levels: first, it led to an inactivation (estimated to 2.3 log); second it led to a spore injury and then to a spore inhibition. This latter effect was assessed by analysing data from a challenge test study carried out with Clostridium sporogenes spores in the foie gras product. The probability of spore recovering after thermal inhibition was estimated to 9.5×10(-8) (corresponding to 7.0 log). The data on the consumption pattern were collected on the French market. The Quantitative Microbiological Risk Assessment (QMRA) model and all the assumptions are reported in detail in the study. The initial contamination of raw materials, effect of thermal treatment on microbial inactivation and spore inhibition were handled mathematically using a probabilistic framework, considering only the variability dimension. The model was implemented in Excel and run through Monte Carlo simulation, using @Risk software. In parallel, epidemiological data collected from the French Institute for Public Health Surveillance during the period 2001-2012 were used to estimate an Appropriate Level Of Protection (ALOP) and then a Food Safety Objective (FSO): ALOP equalled to 2.5×10(-3) illnesses per million inhabitant per year, FSO equalled to 1.4×10(-9) foie gras portions containing C. botulinum spore (expressed in decimal logarithm, FSO=-8.9). The QMRA model output values were smaller, but on the same order of magnitude as these two figures: 8.0×10(-4) illnesses per million inhabitants per year, and, 4.5×10(-10) (-9.3 log) foie gras portions containing C

Rabbit alveolar macrophages were cultured in an environment conducive to the secretion of both reactive oxygen and proteinases, so that the relative importance of proteolytic and oxidative inactivation of alpha 1-proteinase inhibitor by alveolar macrophages could be evaluated. The inactivation of alpha 1-proteinase inhibitor was proportional to its proteolysis, and there was no detectable inactivation in the absence of proteolysis. Although the live macrophages were capable of secreting reactive oxygen, they did not inactivate alpha 1-proteinase inhibitor by oxidation. The inactivation of alpha 1-proteinase inhibitor by proteolysis was proportional to the secretion of elastinolytic activity by the alveolar macrophages. The inability of the alveolar macrophages to oxidize alpha 1-proteinase inhibitor was attributed to the methionine in the macrophages, in secreted proteins, and in the culture medium competing for oxidants. The data suggest that proteolytic inactivation of alpha 1-proteinase inhibitor may be important in vivo and that the methionine concentration in vivo may protect alpha 1-proteinase inhibitor from significant oxidative inactivation. Images PMID:2989330

Porcine epidemic diarrhea virus (PEDV) replicates in the cytoplasm of infected cells, but its nucleocapsid (N) protein localizes specifically to the nucleolus. The mechanism of nuclear translocation, and whether N protein associates with particular nucleolar components, is unknown. In this study, we confirm that a nucleolar phosphoprotein nucleophosmin (NPM1) interacts and co-localizes with the N protein in the nucleolus. In vitro binding studies indicated that aa 148–294 of N and aa 118–188 of NPM1 were required for binding. Interestingly, N protein importation into the nucleolus is independent of the ability of NPM1 to shuttle between the nucleus and the cytoplasm. Furthermore, overexpression of NPM1 promoted PEDV growth, while knockdown of NPM1 suppressed PEDV growth. In addition, binding of N protein to NPM1 protects it from proteolytic degradation by caspase-3, leading to increased cell survival. Taken together, our studies demonstrate a specific interaction of the N protein with the host cell protein NPM1 in the nucleolus. The results suggest potential linkages among viral strategies for the regulation of cell survival activities, possibly through an interaction of N protein with NPM1 which prevents its proteolytic cleavage and enhances cell survival, thus ultimately promoting the replication of PEDV. PMID:28045037

Immunosuppression induced by measles virus (MV) is associated with unresponsiveness of peripheral blood lymphocytes (PBL) to mitogenic stimulation ex vivo and in vitro. In mixed lymphocyte cultures and in an experimental animal model, the expression of the MV glycoproteins on the surface of UV-inactivated MV particles, MV-infected cells, or cells transfected to coexpress the MV fusion (F) and the hemagglutinin (H) proteins was found to be necessary and sufficient for this phenomenon. We now show that MV fusion-inhibitory peptides do not interfere with the induction of immunosuppression in vitro, indicating that MV F-H-mediated fusion is essentially not involved in this process. Proteolytic cleavage of MV F0 protein by cellular proteases, such as furin, into the F1-F2 subunits is, however, an absolute requirement, since (i) the inhibitory activity of MV-infected BJAB cells was significantly impaired in the presence of a furin-inhibitory peptide and (ii) cells expressing or viruses containing uncleaved F0 proteins revealed a strongly reduced inhibitory activity which was improved following trypsin treatment. The low inhibitory activity of effector structures containing mainly F0 proteins was not due to an impaired F0-H interaction, since both surface expression and cocapping efficiencies were similar to those found with the authentic MV F and H proteins. These results indicate that the fusogenic activity of the MV F-H complexes can be uncoupled from their immunosuppressive activity and that the immunosuppressive domains of these proteins are exposed only after proteolyticactivation of the MV F0 protein. PMID:10644371

Tetraspanins are a family of ubiquitously expressed and conserved proteins, which are characterized by four transmembrane domains and the formation of a short and a large extracellular loop (LEL). Through interaction with other tetraspanins and transmembrane proteins such as growth factors, receptors and integrins, tetraspanins build a wide ranging and membrane spanning protein network. Such tetraspanin-enriched microdomains (TEMs) contribute to the formation and stability of functional signaling complexes involved in cell activation, adhesion, motility, differentiation, and malignancy. There is increasing evidence showing that the tetraspanins also regulate the proteolysis of the amyloid precursor protein (APP) by physically interacting with the APP secretases. CD9, CD63, CD81, Tspan12, Tspan15 are among the tetraspanins involved in the intracellular transport and in the stabilization of the gamma secretase complex or ADAM10 as the major APP alpha secretase. They also directly regulate, most likely in concert with other tetraspanins, the proteolytic function of these membrane embedded enzymes. Despite the knowledge about the interaction of tetraspanins with the secretases not much is known about their physiological role, their importance in Alzheimer's Disease and their exact mode of action. This review aims to summarize the current knowledge and open questions regarding the biology of tetraspanins and the understanding how these proteins interact with APP processing pathways. Ultimately, it will be of interest if tetraspanins are suitable targets for future therapeutical approaches. PMID:28066176

The glycosylphosphatidylinositol (GPI)-anchored proteoglycan Cripto binds Nodal and its type I receptor Alk4 to activate Smad2,3 transcription factors, but a role during Nodal precursor processing has not been described. We show that Cripto also binds the proprotein convertases Furin and PACE4 and localizes Nodal processing at the cell surface. When coexpressed as in early embryonic cells, Cripto and uncleaved Nodal already associated during secretion, and a Cripto-interacting region in the Nodal propeptide potentiated the effect of proteolytic maturation on Nodal signalling. Disruption of the trans-Golgi network (TGN) by brefeldin A blocked secretion, but export of Cripto and Nodal to the cell surface was not inhibited, indicating that Nodal is exposed to extracellular convertases before entering the TGN/endosomal system. Density fractionation and antibody uptake experiments showed that Cripto guides the Nodal precursor in detergent-resistant membranes to endocytic microdomains marked by GFP-Flotillin. We conclude that Nodal processing and endocytosis are coupled in signal-receiving cells.

A long-chain amino acid containing dipeptide has been found to form a hydrogel in phosphate buffer whose pH ranges from 6.0 to 8.8. The hydrogel formed at pH 7.46 has been characterized by small-angle X-ray scattering (SAXS), wide-angle powder X-ray diffraction (PXRD), Fourier transform infrared (FT-IR) spectroscopy, field-emission scanning electron microscopy (FE-SEM), high-resolution transmission electron microscopy (HR-TEM) imaging and rheological analyses. The microscopic imaging studies suggest the formation of a nanofibrillar three-dimensional (3D) network for the hydrogel. As observed visually and confirmed rheologically, the hydrogel at pH 7.46 exhibits thixotropy. This thixotropic property can be exploited to inject the peptide. Furthermore, the hydrogel exhibits remarkable antibacterial activity against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, which are responsible for many common diseases. The hydrogel has practical applicability due to its biocompatibility with human red blood cells and human fibroblast cells. Interestingly, this hydrogel shows high resistance toward proteolytic enzymes, making it a new potential antimicrobial agent for future applications. It has also been observed that a small change in molecular structure of the gelator peptide not only turns the gelator into a nongelator molecule under similar conditions, but it also has a significant negative impact on its bactericidal character.

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA. PMID:17686855

Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.

Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolyticactivities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

Enzymes and other bio-macromolecules are not only sensitive to physical parameters such as pH, temperature and solute composition but also to water activity. A universally instructive way to vary water activity is the addition of osmotically active but otherwise inert solvents which also mimic the condition of an intercellular milieu. In the present contribution, the role of hydration on the functionality of a proteolytic enzyme α-chymotrypsin (CHT) is investigated by modulating the water activity with the addition of polyethylene glycols (PEG with an average molecular weight of 400). The addition of PEG increases the affinity of the enzyme to its substrate, however, followed by a decrease in the turnover number (k(cat)). Energetic calculations show that entrance path for the substrate is favoured, whereas the exit channel is restricted with increasing concentration of the crowding agent. This decrease is attributed to the thinning of the hydration shell of the enzyme due to the loss of critical water residues from the hydration surface of the enzyme as evidenced from volumetric and compressibility measurements. The overall secondary and tertiary structures of CHT determined from far-UV and near-UV circular dichroism (CD) measurements show no considerable change in the studied osmotic stress range. From kinetic and equilibrium data, we calculate 115 ± 30 numbers of water molecules to be altered during the enzymatic catalysis of CHT. Spectroscopic observation of water relaxation and rotational dynamics of ANS-CHT complex at various concentrations of the osmoting agent also support the dehydration of the hydration layer. Such dehydration/hydration processes during turnover imply a significant contribution of solvation to the energetics of the conformational changes.

The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5–7, but the aspartic proteases were inactive across a pH range of 3–10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi. PMID:24401858

Recently, utilization of the enzyme Chondroitinase ABC I (cABC I) has received considerable attention in treatment of spinal cord injury. cABC I removes chondroitin sulfate proteoglycans which are inhibitory to axon growth and enhances nerve regeneration. Therefore, determination of cABC I resistance to proteolysis and oxidation provides valuable information for optimizing its clinical application. In this work, proteolytic stability of cABC I to trypsin and chymotrypsin as well as its oxidative resistance to H2O2 was measured. Moreover, the effect of cosolvents glycerol, sorbitol and trehalose on cABC I proteolytic and oxidative stability was determined. The results indicated that cABC I is highly susceptible to proteolysis and oxidation. Comparison of proteolytic patterns demonstrated a high degree of similarity which confirmed the exposure of specific regions of cABC I to proteolysis. However, proteolytic degradation was significantly reduced in the presence of cosolvents. In addition, cosolvents decreased the rate of both cABC I proteolytic and oxidative inactivation. Notably, the degree of stabilization provided by these cosolvents varied greatly. These findings indicated the high potential of cosolvents in protein stabilization to proteolysis and oxidative inactivation.

Over the past decade, important roles for the 84–88 kDa Group VIA Ca2+-independent phospholipase A2 (iPLA2β) in various organs have been described. We demonstrated that iPLA2β participates in insulin secretion, insulinoma cells and native pancreatic islets express full-length and truncated isoforms of iPLA2β, and certain stimuli promote perinuclear localization of iPLA2β. To gain a better understanding of its mobilization, iPLA2β was expressed in INS-1 cells as a fusion protein with EGFP, enabling detection of subcellular localization of iPLA2β by monitoring EGFP fluorescence. Cells stably-transfected with fusion protein expressed nearly 5-fold higher catalytic iPLA2β activity than control cells transfected with EGFP cDNA alone, indicating that co-expression of EGFP does not interfere with manifestation of iPLA2β activity. Dual fluorescence monitoring of EGFP and organelle Trackers combined with immunoblotting analyses revealed expression of truncated iPLA2β isoforms in separate subcellular organelles. Exposure to secretagogues and induction of ER stress are known to activate iPLA2β in β-cells and we find here that these stimuli promote differential localization of iPLA2β in subcellular organelles. Further, mass spectrometric analyses identified iPLA2β variants from which N-terminal residues were removed. Collectively, these findings provide evidence for endogenous proteolytic processing of iPLA2β and redistribution of iPLA2β variants in subcellular compartments. It might be proposed that in vivo processing of iPLA2β facilitates its participation in multiple biological processes. PMID:20132906

Vip3 proteins have been described to be secreted by Bacillus thuringiensis during the vegetative growth phase and to display a broad insecticidal spectrum against lepidopteran larvae. Vip3Aa protoxin has been reported to be significantly more toxic to Spodoptera frugiperda than to Spodoptera exigua and differences in the midgut processing have been proposed to be responsible. In contrast, we have found that Vip3Ae is essentially equally toxic against these two species. Proteolysis experiments were performed to study the stability of Vip3A proteins to peptidase digestion and to see whether the differences found could explain differences in toxicity against these two Spodoptera species. It was found that activation of the protoxin form and degradation of the 62kDa band took place at lower concentrations of trypsin when using Vip3Aa than when using Vip3Ae. The opposite effect was observed for chymotrypsin. Vip3Aa and Vip3Ae protoxins were effectively processed by midgut content extracts from the two Spodoptera species and the proteolyticactivation did not produce a peptidase resistant core under these in vitro conditions. Digestion experiments performed with S. frugiperda chromatography-purified digestive serine peptidases showed that the degradation of the Vip3A toxins active core is mainly due to the action of cationic chymotrypsin-like peptidase. Although the digestion patterns of Vip3A proteins do not always correlate with toxicity, the peptidase stability of the 62kDa core is in agreement with intraspecific differences of toxicity of the Vip3Aa protein.

Proteinase cascades are part of the basic machinery of neuronal death pathways. Neuronal cathepsin B (CatB), a typical cysteine lysosomal protease, plays a critical role in neuronal death through lysosomal leakage or excessive autophagy. On the other hand, much attention has been paid to microglial CatB in neuronal death. We herein show the critical role of proteolytic relay through microglial CatB and CatE in the polarization of microglia/macrophages in the neurotoxic phenotype, leading to hypoxia/ischemia (HI)-induced hippocampal neuronal damage in neonatal mice. HI caused extensive brain injury in neonatal wild-type mice, but not in CatB(-/-) mice. Furthermore, HI-induced polarization of microglia/macrophages in the neurotoxic phenotype followed by the neuroprotective phenotype in wild-type mice. On the other hand, microglia/macrophages exhibited only the early and transient polarization in the neuroprotective phenotype in CatB(-/-) mice. CA-074Me, a specific CatB inhibitor, significantly inhibited the neuronal death of primary cultured hippocampal neurons induced by the conditioned medium from cultured microglia polarized in the neurotoxic phenotype. Furthermore, CA-074Me prevented the activation of nuclear factor-κB (NF-κB) in cultured microglia by inhibiting autophagic inhibitor of κBα degradation following exposure to oxygen-glucose deprivation. Rather surprisingly, CatE increased the CatB expression after HI by the liberation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from microglia through the proteasomal pathway. A significant increase in CatB and CatE levels was found exclusively in microglia/macrophages after HI. Thus, a proteolytic relay through the early CatE/TRAIL-dependent proteosomal and late CatB-dependent autophagic pathways for NF-κB activation may play a critical role in the polarization of microglia/macrophages in the neurotoxic phenotype. Significance statement: Proteinase cascades are part of the basic

Relationships of the somatic antigens among Clostridium botulinum strains have been investigated by tube agglutination and agglutinin absorption tests. Results revealed a relationship by which strains of C. botulinum are grouped by their proteolytic capacity rather than by the type of specific toxin produced. Thus, C. botulinum type E and its nontoxigenic variants, which are nonproteolytic, share common somatic antigens with the nonproteolytic strains of types B and F. Absorption of antiserum of a strain of any one type with antigen of any of the others removes the antibody to all three types. In the same manner, C. botulinum type A shares somatic antigens with the proteolytic strains of types B and F, and absorption of any one antiserum with an antigen of either of the other two types removes the antibody to all three types. Partial cross-agglutination of C. sporogenes, C. tetani, and C. histolyticum with the somatic antisera of the proteolytic group was also observed. PMID:4927406

Proteolytic equilibria of vanillic acid in aqueous solutions were studied using electronic spectroscopy. The pH ranges for anionic, dianionic, cationic, and neutral forms of vanillic acid in the ground and excited states were determined. The electron density distribution on atoms in the proteolytic forms was determined using quantum-chemistry methods. The anion formed as a result of dissociation of the carboxylic acid. The dianion formed in the presence of two and more equivalents of alkali as a result of proton loss from the phenol and carboxylic acid. The vanillic acid cation formed via protonation of the carbonyl oxygen. Differences in spectral features of the proteolytic forms in the ground and excited states were observed.

Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significant improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.

Knockout mice are the gold standard to probe for the role of a specific protease within the interacting network of proteases, substrates, and inhibitors. This proteolytic network, or protease web, determines cell signaling and organ homeostasis. Therefore, protease deficiency or inhibition is intrinsically tied to alterations within this network, always leading to new molecular phenotypes, which define susceptibility of an organ to disease. Furthermore, recent hints, mainly from research on matrix metalloproteinases, about the impact of the protease web on inter-organ signaling molecules suggest the existence of a proteolytic internet of communicating local organ- or molecular polymorphism-specific networks, thereby defining homeostasis and disease susceptibility in the whole organism.

Bone morphogenetic protein-7 (BMP-7) is synthesized as a precursor that requires proteolytic cleavage of the propeptide by proprotein convertases (PCs) for its functional activity. A high-level expression of BMP-7 in CHO cells (CHO-BMP-7) resulted in secretion of a mixture of inactive precursor and active BMP-7. In an effort to achieve efficient processing of BMP-7 in CHO cells, PCs responsible for cleavage of the precursors in CHO cells were characterized. Analysis of the mRNA expression levels of four PCs (furin, PACE4, PC5/6, and PC7) revealed that only furin and PC7 genes are expressed in CHO-BMP-7 cells. Specific inhibition of the PCs by hexa-D-arginine (D6R) or decanoyl-RVKR-chloromethyl ketone (RVKR-CMK) further revealed that furin is mainly responsible for the proteolytic processing of BMP-7. To identify a more efficient PC for BMP-7 processing, the four PC genes were transiently expressed in CHO-BMP-7 cells, respectively. Among these, PC5/6 was found to be the most efficient in BMP-7 processing. Stable overexpression of PC5/6ΔC, a secreted form of PC5/6, significantly improved mature BMP-7 production in CHO-BMP-7 cells. When the maximum BMP-7 concentration was obtained in the culture of CHO-BMP-7 cells, approximately 88% of BMP-7 was unprocessed. In contrast, no precursor was found in the culture of PC5/6ΔC-overexpressing cells (clone #97). Furthermore, the in vitro biological activity of the mature BMP-7 from PC5/6ΔC-overexpressing cells was comparable to that from CHO-BMP-7 cells. Taken together, the present results indicate that overexpression of PC5/6ΔC in CHO-BMP-7 cells is an efficient means of increasing the yield of BMP-7.

Paper-based assays are a promising diagnostic format for point-of-care applications, field deployment, and other low-resource settings. To date, the majority of efforts to integrate nanomaterials with paper-based assays have utilized gold nanoparticles. Here, we show that semiconductor quantum dots (QDs), in combination with Förster resonance energy transfer (FRET), are also suitable nanomaterials for developing paper-based assays. Paper fibers were chemically modified with thiol ligands to immobilize CdSeS/ZnS QDs, the QDs were self-assembled with dye-labeled peptides to generate efficient FRET, and steady-state and fluorescence lifetime imaging microscopy (FLIM) were used for characterization. Peptides were selected as substrates for three different proteases and a series of kinetic assays for proteolyticactivity was carried out, including multiplexed assays and pro-enzyme activation assays. Quantitative results were obtained within 5-60 min at levels as low as 1-2 nM of protease. These assays were possible using simple optical readout platforms that did not negate the low cost, ease of use, and overall accessibility advantages of paper-based assays. A violet light-emitting diode (LED) excitation source and color imaging with either a digital camera, consumer webcam, or smartphone camera were sufficient for analysis on the basis of a red/green color intensity ratio. At most, a universal serial bus (USB) connection to a computer was required and the instrumentation cost orders of magnitude less than that typically utilized for in vitro bioanalyses with QDs. This work demonstrates that QDs are valuable probes for developing a new generation of paper-based diagnostics.

Our aim was to investigate the role of oxidative stress in elastase-induced pulmonary emphysema. C57BL/6 mice were subjected to pancreatic porcine elastase (PPE) instillation (0.05 or 0.5 U per mouse, i.t.) to induce pulmonary emphysema. Lungs were collected on days 7, 14, and 21 after PPE instillation. The control group was sham injected. Also, mice treated with 1% aminoguanidine (AMG) and inducible NO synthase (iNOS) knockout mice received 0.5 U PPE (i.t.), and lungs were analyzed 21 days after. We performed bronchoalveolar lavage, biochemical analyses of oxidative stress, and lung stereology and morphometry assays. Emphysema was observed histologically at 21 days after 0.5 U PPE treatment; tissues from these mice exhibited increased alveolar linear intercept and air-space volume density in comparison with the control group. TNF-α was elevated at 7 and 14 days after 0.5 U PPE treatment, concomitant with a reduction in the IL-10 levels at the same time points. Myeloperoxidase was elevated in all groups treated with 0.5 U PPE. Oxidative stress was observed during early stages of emphysema, with increased nitrite levels and malondialdehyde and superoxide dismutase activity at 7 days after 0.5 U PPE treatment. Glutathione peroxidase activity was increased in all groups treated with 0.5 U PPE. The emphysema was attenuated when iNOS was inhibited using 1% AMG and in iNOS knockout mice. Furthermore, proteolytic stimulation by PPE enhanced the expression of nitrotyrosine and iNOS, whereas the PPE+AMG group showed low expression of iNOS and nitrotyrosine. PPE stimulus also induced endothelial (e) NOS expression, whereas AMG reduced eNOS. Our results suggest that the oxidative and nitrosative stress pathways are triggered by nitric oxide production via iNOS expression in pulmonary emphysema.

Facile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However, electrochemical detection of proteases, such as botulinum neurotoxin type E (BoNT/E), that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase, L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to generate isoleucine-AN. Subsequently, LAP cleaves a bond between isoleucine and AN to liberate a free electroactive AN species. The liberated AN participates in electrochemical-chemical-chemical (ECC) redox cycling involving Ru(NH3)6(3+), AN, and a reducing agent, which allows a high signal amplification. Electrochemical detection is carried out without surface modification of indium-tin oxide electrodes. We show that dithiothreitol is beneficial for enhancing the enzymatic activity of BoNT/E-LC and also for achieving a fast ECC redox cycling. An incubation temperature of 37°C and the use of phosphate buffered saline (PBS) buffer resulted in optimal signal-to-background ratios for efficient BoNT/E detection. BoNT/E-LC could be detected at concentrations of approximately 2.0 pg/mL, 0.2, and 3 ng/mL after 4h, 2h, and 15 min incubation in PBS buffer, respectively, and approximately 0.3 ng/mL after 2-h incubation in bottled water. The method developed could be applied in fast, sensitive, and selective detection of any protease that cleaves a peptide bond between two specific amino acid residues.

Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

One critical step in porcine reproductive and respiratory syndrome virus (PRRSV) replication is the proteolytic processing of the ORF1 polyprotein (replicase). The replicase polyprotein is generally believed to be processed to generate at least 12 smaller nonstructural proteins (nsps) involved in r...

The nsp2 replicase protein of porcine reproductive and respiratory syndrome virus (PRRSV) was recently demonstrated to be processed from its precursor by the PL2 protease at or near the G1196|G1197 dipeptide in transfected CHO cells. Here, the proteolytic cleavage of PRRSV nsp2 was further investiga...

Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis.

Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum (C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic C. botulinum species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic C. botulinum types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic C. botulinum types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains. PMID:15746333

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum (C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic C. botulinum species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic C. botulinum types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic C. botulinum types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.

The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples.

overexpress hK2 to generate models that can be used for in vivo testing. Studies with Human Breast Cancer extracts Our original intention was to...microsomes for detection by fluorescence based cell sorting. Once we have optimized the system we will begin to analyze extracts from breast...tested for hydrolysis in breast cancer ECF extracts to determine best peptide sequence for further development 10 DAMD17-03-1-0304 P.I. Denmeade

hK2 to generate models that can be used for in vivo testing. Studies with Human Breast Cancer extracts Our original intention was to incubate this...detection by fluorescence based cell sorting. Once we have optimized the system we will begin to analyze extracts from breast tissues and conditioned...fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 247:954-958, 1990. 27. Chagas JR, Juliano L, Prado ES

Effective clearance of apoptotic cells by phagocytes prevents the release of intracellular alarmins and manifestation of autoimmunity. This prompt efferocytosis is complemented by intracellular proteolytic degradation that occurs within the apoptotic cells and in the efferosome of the phagocytes. Although the role of extracellular proteases in apoptotic cells clearance is unknown, the strong association of congenital C1s deficiency with Systemic Lupus Erythematosus highlights the protective nature that this extracellular protease has against autoimmunity. The archetypical role of serine protease C1s as the catalytic arm of C1 complex (C1qC1r2C1s2) involve in the propagation of the classical complement pathway could not provide the biological basis for this association. However, a recent observation of the ability of C1 complex to cleave a spectrum of intracellular cryptic targets exposed during apoptosis provides a valuable insight to the underlying protective mechanism. High-mobility group box 1 (HMGB1), an intracellular alarmin that is capable of inducing the formation of antinuclear autoantibodies and causes lupus-like conditions in mice, is identified as a novel potential target by bioinformatics analysis. This is verified experimentally with C1s, both in its purified and physiological form as C1 complex, cleaving HMGB1 into defined fragments of 19 and 12 kDa. This cleavage diminishes HMGB1 ability to enhance lipopolysaccharide mediated pro-inflammatory cytokines production from monocytes, macrophages and dendritic cells. Further mass spectrometric analysis of the C1 complex treated apoptotic cellular proteins demonstrated additional C1s substrates and revealed the complementary role of C1s in apoptotic cells clearance through the proteolytic cleavage of intracellular alarmins and autoantigens. C1 complex may have evolved as, besides the bacteriolytic arm of antibodies in which it activates the complement cascade, a tissue renewal mechanism that reduces the

Despite their central importance in cell metabolism, little is known about proteases in marine phytoplankton. Caseinolytic and leucine aminopeptidase (LAP) activities was surveyed in log-phase cultures of the chlorophyte Dunaliella tertiolecta Butcher, the diatom Thalassiosira weissflogii Fryxell et Hasle, the chrysophyte Isochrysis galbana Parke, the coccolithophorid Emiliania huxleyi Hay et Mohler, and the cyanobacterium Synechococcus sp. LAP activity was very low at pH < 6 and peaked between pH 7.5 and 8.5 in all species, whereas caseinolytic activity in most species showed only minor peaks in the pH 4-5 range and broad maxima above pH 8. Acidic vacuolar proteases apparently represented only a small fraction of total protease activity. Attempts to classify protease using selective inhibitors were inconclusive. Caserinolytic activities were remarkably stable. Casein zymograms were used to identify >200-and <20-kDa proteases in homogenates of log-phase T. weissflogii; only the smaller protease was found in D. tertiolecta. Antibodies in the ATPase subunit (C) of the conserved, chloroplastic Clp protease from Pisum cross-reacted with proteins in Synechococcus, D. tertiolecta, and I. galbana, but no cross-reactions were found for any species with antibodies against the ClpP subunit from either E. coli or Nicotiana. Our results show that phytoplankton contain a diverse complement of proteases with novel characteristics. 46 refs., 6 figs., 1 tab.

Extracellular products (ECP) produced by Vibrio anguillarum strain M3 originally isolated from diseased flounder ( Paralichthys olivaceus) were prepared. ECP of M3 showed gelatinase, casinase, amylase and haemolytic activity on agarose plates. High protease activity against azocasin was detected. Bacterium M2 showed highest growth and protease activity at 25°C. The protease present in ECP showed maximal activity at pH 8 and 55°C; was completely inactivated by application of 80°C heat for 30 min; was completely inhibited by EDTA and HgCl2, and was partially inhibited by PMSF, SDS, MnCl2 and iodoacetic acid; but not inhibited by CaCl2 and MgCl2. The ECP was toxic to flounder fish at LD50 values of 3.1 μg protein/g body weight. The addition of HgCl2 and application of heat at 50°C decreased the lethal toxicity of ECP. When heated at 100°C, ECP lethality to flounder was completely inhibited. After intramuscular injection of ECP into flounder, it showed evident histopathological changes including necrosis of muscle, extensive deposition of haemosiderin in the spleen, dilated blood vessels congested with numerious lymphocytes in the liver. These results showed that ECP protease was a lethal factor produced by the bacterium V. anguillarum M3.

produces early and sustained increases in calpain and caspae -3 activity that vary depending upon the brain regions studies. (2) TBI can produce...different brain regions. Changes may be differentially localized in subcellular fractions. (4) Increased magnitudes and duration of calpain and caspae -3

Pseudomonas aeruginosa, a Gram-negative bacterium, is a significant opportunistic pathogen associated with skin and soft tissue infections, nosocomial pneumonia and sepsis. In addition, it can chronically colonize the lungs of cystic fibrosis (CF) patients. Overproduction of the exopolysaccharide called alginate provides P. aeruginosa with a selective advantage and facilitates survival in the CF lung. The in vitro phenotype of alginate overproduction observed on solid culture media is referred to as mucoid. Expression of the alginate machinery and biosynthetic enzymes are controlled by the extracytoplasmic sigma factor, σ(22) (AlgU/T). The key negative regulator of both σ(22) activity and the mucoid phenotype is the cognate anti-sigma factor MucA. MucA sequesters σ(22) to the inner membrane inhibiting the sigma factor's transcriptional activity. The well-studied mechanism for transition to the mucoid phenotype is mutation of mucA, leading to loss of MucA function and therefore activation of σ(22) . Recently, regulated intramembrane proteolysis (RIP) has been recognized as a mechanism whereby proteolysis of the anti-sigma factor MucA leads to active σ(22) allowing P. aeruginosa to respond to environmental stress conditions by overproduction of alginate. The goal of this review is to illuminate the pathways leading to RIP that have been identified and proposed.

This study was undertaken to assess the effect of somatic cell count in ewe milk on i) composition and hygienic traits; ii) plasmin, cathepsin and elastase activities; iii) leukocyte differential count; iv) renneting parameters. Individual ewe milk samples were grouped according to somatic cell count (SCC) into five classes: SC300 (<300 000 cells/ml), SC500 (from 301 000 to 500 000 cells/ml), SC1000 (from 501 000 to 1 000 000 cells/ml), SC2000 (from 1 001 000 to 2 000 000 cells/ml) and SC>2000 (>2 001 000 cells/ml). Individual milk samples were analysed for pH, chemical composition, microbial features, indigenous proteolytic enzymes, differential leukocyte population, and renneting parameters. Milk yield, lactose, protein, non casein nitrogen, microbial features were affected by SCC level. Plasmin and elastase activities were the highest in samples with more than 1 000 000 cells/ml; plasmin had intermediate values in samples with 300 000 to 1 000 000 cells/ml and the lowest in samples with less than 300 000 cells/ml of milk. Cathepsin D showed significantly lower values in SC300 and SC1000 classes than in SC500, SC2000 and SC>2000 classes. The highest percentages of lymphocyte were found in samples with less than 1 000 000 cells/ml, while the highest levels of polymorphonuclear leukocyte were found in samples with more than 1 000 000 cells/ml of milk. Longer clotting time was found in SC>2000 samples, while reduced clot firmness was observed in SC500 and SC>2000 samples. Results on milk yield and on compositional parameters evidenced an impairment of udder efficiency in ewe milk samples starting from 300 000 cells/ml. Plasmin activity in milk can be considered as a marker of the synthetic and secreting ability of the mammary gland; furthermore plasmin and elastase were consistent with the health status of the udder. Finally cathepsin D played a role in the worsening of renneting properties of ewe milk.

HIV-1 has been the target of intensive research at the molecular and biochemical levels for >25 years. Collectively, this work has led to a detailed understanding of viral replication and the development of 24 approved drugs that have five different targets on various viral proteins and one cellular target (CCR5). Although most drugs target viral enzymatic activities, our detailed knowledge of so much of the viral life cycle is leading us into other types of inhibitors that can block or disrupt protein-protein interactions. Viruses have compact genomes and employ a strategy of using a small number of proteins that can form repeating structures to enclose space (i.e. condensing the viral genome inside of a protein shell), thus minimizing the need for a large protein coding capacity. This creates a relatively small number of critical protein-protein interactions that are essential for viral replication. For HIV-1, the Gag protein has the role of a polyprotein precursor that contains all of the structural proteins of the virion: matrix, capsid, spacer peptide 1, nucleocapsid, spacer peptide 2, and p6 (which contains protein-binding domains that interact with host proteins during budding). Similarly, the Gag-Pro-Pol precursor encodes most of the Gag protein but now includes the viral enzymes: protease, reverse transcriptase (with its associated RNase H activity), and integrase. Gag and Gag-Pro-Pol are the substrates of the viral protease, which is responsible for cleaving these precursors into their mature and fully active forms (see Fig. 1A).

Strain SR 394 of Erwinia carotovora (Ecc) produced proteases constitutively in all media tested. Growth of Ecc and production of protease were enhanced significantly by the presence of poetic materials and/or plant call walls in the test media. After electrofocusing, one major and one minor protease bands, at PI 4.8 and PI 5.1, respectively, were detected. Only one band of 43 kDa was detected on SDS gels. Only one protease band was detected in SDS gels of infected plant extracts. This protease was purified to homogeneity. It in a highly thermostable metal protease; it degrades gelatin, soluble collagen and hide powderazure, shows weak activity on casein and azocasein, but does not degrade insoluble collagen or elastin.

There is currently no effective treatment for multiorgan failure following shock other than supportive care. A better understanding of the pathogenesis of these sequelae to shock is required. The intestine plays a central role in multiorgan failure. It was previously suggested that bacteria and their toxins are responsible for the organ failure seen in circulatory shock, but clinical trials in septic patients have not confirmed this hypothesis. Instead, we review here evidence that the digestive enzymes, synthesized in the pancreas and discharged into the small intestine as requirement for normal digestion, may play a role in multiorgan failure. These powerful enzymes are nonspecific, highly concentrated, and fully activated in the lumen of the intestine. During normal digestion they are compartmentalized in the lumen of the intestine by the mucosal epithelial barrier. However, if this barrier becomes permeable, e.g. in an ischemic state, the digestive enzymes escape into the wall of the intestine. They digest tissues in the mucosa and generate small molecular weight cytotoxic fragments such as unbound free fatty acids. Digestive enzymes may also escape into the systemic circulation and activate other degrading proteases. These proteases have the ability to clip the ectodomain of surface receptors and compromise their function, for example cleaving the insulin receptor causing insulin resistance. The combination of digestive enzymes and cytotoxic fragments leaking into the central circulation causes cell and organ dysfunction, and ultimately may lead to complete organ failure and death. We summarize current evidence suggesting that enteral blockade of digestive enzymes inside the lumen of the intestine may serve to reduce acute cell and organ damage and improve survival in experimental shock.

Tissue engineering aims at developing the necessary technological strategies for replacement or regeneration tissues. However, the number of cells required is much greater than the number obtained from a cell source. Expanding the cells' number in cell culture for a long period is required until the necessary amount of cells is obtained. While in culture, cells often go unwanted differentiation. Little attention has been given to the use of proteolytic enzymes in cell passage. Review the importance of extracellular matrix and surface proteins for cell behavior and the possible mechanisms of cellular changes that may occur due to the use of proteolytic enzymes is an essential issue. Possible alternative to replace these enzymes in cell passage has also been developed. PMID:28031901

Proteolytic processing events are essential to physiological processes such as reproduction, development, and host responses, as well as regulating proteins in cancer; therefore, there is a significant need to develop robust approaches for characterizing such events. The current mass spectrometry (MS)-based proteomics techniques employs a “bottom-up” strategy, which does not allow for identification of different proteolytic proteins since the strategy measures all the small peptides from any given protein. The aim of this development is to enable the effective identification of specific proteolytic fragments. The protocol utilizes an acetylation reaction to block the N-termini of a protein, as well as any lysine residues. Following digestion, N-terminal peptides are enriched by removing peptides that contain free amines, using amine-reactive silica-bond succinic anhydride beads. The resulting enriched sample has one N-terminal peptide per protein, which reduces sample complexity and allows for increased analytical sensitivity compared to global proteomics.1 We initially compared the peptide identification and efficiency of blocking lysine using acetic anhydride (a 42 Da modification) or propionic anhydride (a 56 Da modification) in our protocol. Both chemical reactions resulted in comparable peptide identifications and *95 percent efficiency for blocking lysine residues. However, the use of propionic anhydride allowed us to distinguish in vivo acetylated peptides from chemically-tagged peptides.2 In an initial experiment using mouse plasma, we were able to identify *300 unique N-termini peptides, as well as many known cleavage sites. This protocol holds potential for uncovering new information related to proteolytic pathways, which will assist our understanding about cancer biology and efforts to identify potential biomarkers for various diseases.

Phosphodiesterase-6 (PDE6) is an essential effector enzyme in vertebrate photoreceptor cells. Mutations in rod and cone PDE6 cause recessive retinitis pigmentosa and achromatopsia, respectively. The mechanisms of missense PDE6 mutations underlying severe visual disorders are poorly understood. To probe these mechanisms, we expressed seven known missense mutants of cone PDE6C in rods of transgenic Xenopus laevis and examined their stability and compartmentalization. PDE6C proteins with mutations in the catalytic domain, H602L and E790K, displayed modestly reduced proteolytic stability, but they were properly targeted to the outer segment of photoreceptor cells. Mutations in the regulatory GAF domains, R104W, Y323N, and P391L led to a proteolytic degradation of the proteins involving a cleavage in the GAFb domain. Lastly, the R29W and M455V mutations residing outside the conserved PDE6 domains produced a pattern of subcellular compartmentalization different from that of PDE6C. Thus, our results suggest a spectrum of mechanisms of missense PDE6C mutations in achromatopsia including catalytic defects, protein mislocalization, or a specific sequence of proteolytic degradation.

Human immunodeficiency virus particles undergo a step of proteolytic maturation, in which the main structural polyprotein Gag is cleaved into its mature subunits matrix (MA), capsid (CA), nucleocapsid (NC) and p6. Gag proteolytic processing is accompanied by a dramatic structural rearrangement within the virion, which is necessary for virus infectivity and has been proposed to proceed through a sequence of dissociation and reformation of the capsid lattice. Morphological maturation appears to be tightly regulated, with sequential cleavage events and two small spacer peptides within Gag playing important roles by regulating the disassembly of the immature capsid layer and formation of the mature capsid lattice. In order to measure the influence of individual Gag domains on lattice stability, we established Förster's resonance energy transfer (FRET) reporter virions and employed rapid kinetic FRET and light scatter measurements. This approach allowed us to measure dissociation properties of HIV-1 particles assembled in eukaryotic cells containing Gag proteins in different states of proteolytic processing. While the complex dissociation behavior of the particles prevented an assignment of kinetic rate constants to individual dissociation steps, our analyses revealed characteristic differences in the dissociation properties of the MA layer dependent on the presence of additional domains. The most striking effect observed here was a pronounced stabilization of the MA-CA layer mediated by the presence of the 14 amino acid long spacer peptide SP1 at the CA C-terminus, underlining the crucial role of this peptide for the resolution of the immature particle architecture.

Studies were done to determine the effect of mastic resin and its essential oil, alone and in conjunction with ethanol, on the growth of proteolytic strains of Clostridium botulinum in media, and on neurotoxin production in challenge studies with English-style crumpets. Preliminary studies, using a spot-on-the-lawn method, indicated that high levels of mastic resin in ethanol ( approximately 8% w/w) were required for complete inhibition of all strains of C. botulinum tested, but mastic resin in ethanol had a greater anti-botulinal effect than ethanol alone. However, only low levels of mastic oil ( approximately 0.3% v/v) were required for inhibition of proteolytic strains of C. botulinum. Both studies showed a strain specific inhibition, with C. botulinum type A strains being more sensitive to mastic resin and its essential oil than type B strains. However, mastic resin in ethanol proved to be more effective when used as a vapor phase inhibitor applied to cotton pads and placed inside inoculated plates than when added directly to media. While both mastic resin and its essential oil inhibited the growth of proteolytic strains of C. botulinum in vitro, they failed to inhibit neurotoxin production in challenge studies with C. botulinum in English-style crumpets.

While trypsin remains the most commonly used protease in mass spectrometry, other proteases may be employed for increasing peptide-coverage or generating overlapping peptides. Knowledge of the accurate specifcity rules of these proteases is helpful for database search tools to detect peptides, and becomes crucial when mass spectrometry is used to discover in vivo proteolytic cleavages. In this study, we use tandem mass spectrometry to analyze the specifcity rules of selected proteases and describe MS- Proteolysis, a software tool for identifying putative sites of in vivo proteolytic cleavage. Our analysis suggests that the specifcity rules for some commonly used proteases can be improved, e.g., we find that V8 protease cuts not only after Asp and Glu, as currently expected, but also shows a smaller propensity to cleave after Gly for the conditions tested in this study. Finally, we show that comparative analysis of multiple proteases can be used to detect putative in vivo proteolytic sites on a proteome-wide scale.

Extracts rich in cysteine proteases obtained from fruits of Pseudananas macrodontes (Pm), Bromelia balansae (Bb), and B. hieronymi (Bh) have previously shown an anti-inflammatory effect on animal models. Given the close relationship between hemostasis and inflammation, it is attractive to investigate therapeutic agents capable of modulating both systems. The aim of this work was to study the effect of Pm, Bb, and Bh on fibrin(ogen) and blood coagulation compared with stem bromelain (Bro). Action on fibrinogen was electrophoretically and spectrophotometrically evaluated, fibrinolytic activity was measured both electrophoretically and by the fibrin plate assay, and the effect on blood coagulation was studied by conventional coagulation tests (PT and APPT). All extracts showed the same proteolytic preference for fibrinogen subunits, that is Aα > Bβ, whereas γ was partially hydrolyzed by 100-fold concentration increase. Unlike Bro, cysteine proteases of Pm, Bb, and Bh increased absorbance at 540 nm of fibrinogen solution, suggesting thrombin-like activity, which was time-dependent and reached maximum values at lower concentration. All extracts showed the same proteolytic preference for fibrin subunits; however Pm, Bb, and Bh showed lower fibrinolytic activity than Bro at the assayed concentrations. Although Bb acted only as anticoagulant, Pm, Bh, and unexpectedly Bro showed dual action on blood coagulation: at low concentration showed procoagulant effect and at high concentration anticoagulant effect. Results reveal new plant species as potential sources of pharmacological agents for the treatment of a wide range of hemostatic disorders as well as to wound healing.

The enteric protozoan parasite Entamoeba histolytica causes amebic colitis through disruption of the mucus layer, followed by binding to and destruction of epithelial cells. However, it is not known whether ameba infections or ameba components can directly affect the enteric nervous system. Analysis of mucosal innervations in the mouse model of cecal amebiasis showed that axon density was diminished to less than 25% of control. To determine whether amebas directly contributed to axon loss, we tested the effect of either E. histolytica secreted products (Eh-SEC) or soluble components (Eh-SOL) to an established coculture model of myenteric neurons, glia, and smooth muscle cells. Neuronal survival and axonal degeneration were measured after 48 h of exposure to graded doses of Eh-SEC or Eh-SOL (10 to 80 μg/ml). The addition of 80 μg of either component/ml decreased the neuron number by 30%, whereas the axon number was decreased by 50%. Cytotoxicity was specific to the neuronal population, since the glial and smooth muscle cell number remained similar to that of the control, and was completely abrogated by prior heat denaturation. Neuronal damage was partially prevented by the cysteine protease inhibitor E-64, showing that a heat-labile protease was involved. E. histolytica lysates derived from amebas deficient in the major secreted protease EhCP5 caused a neurotoxicity similar to that of wild-type amebas. We conclude that E. histolytica infection and ameba protease activity can cause selective damage to enteric neurons.

Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aβ), the main pathological component of Alzheimer’s disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bβ-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions. PMID:27437944

Phytoplasmas are insect-borne plant pathogenic bacteria that alter host morphology. TENGU, a small peptide of 38 residues, is a virulence factor secreted by phytoplasmas that induces dwarfism and witches’ broom in the host plant. In this study, we demonstrate that plants process TENGU in order to generate small functional peptides. First, virus vector-mediated transient expression demonstrated that the amino-terminal 11 amino acids of TENGU are capable of causing symptom development in Nicotiana benthamiana plants. The deletion of the 11th residue significantly diminished the symptom-inducing activity of TENGU, suggesting that these 11 amino acids constitute a functional domain. Second, we found that TENGU undergoes proteolytic processing in vitro, generating peptides of 19 and 21 residues including the functional domain. Third, we observed similar processing of TENGU in planta, and an alanine substitution mutant of TENGU, for which processing was compromised, showed reduced symptom induction activity. All TENGU homologs from several phytoplasma strains possessed similar symptom induction activity and went through processing, which suggests that the processing of TENGU might be related to its function. PMID:23784461

The interaction between tyrosine-decarboxylase and proteolyticactivities of a Lactobacillus curvatus and Staphylococcus xylosus, respectively, on biogenic amine production during the ripening and the storage of dry fermented sausages was investigated. Water content, pH, proteolysis parameters, microbial counts, and biogenic amine contents were monitored in spontaneously and starter fermented sausages. The use of proteolytic staphylococci as starter resulted in a higher content of non-protein nitrogen and total free amino acids. Tyramine was the main amine produced in all batches. However, tyrosine-decarboxylase activity of the L. curvatus starter strain was weak and yielded lower amounts of tyramine than those produced by the wild mioroflora in the control batch. Association between tyramine production and proteolysis could only be established in a defectively dried batch. Putrescine and cadaverine accumulation was efficiently reduced in the starter-mediated fermentation, in agreement with the lower development of enterobacteria. Phenylethylamine and tryptamine were only detected in the spontaneously fermented sausages, while histamine, spermine and spermidine did not vary during the ripening. Biogenic amine levels and related parameters showed significant changes during the storage of dry sausages depending on the temperature and the batch. As a general rule, changes in the pH, proteolysis, microbial counts, and biogenic amine contents were stronger at 19 degrees C than at 4 degrees C. The results suggest that refrigeration would be advisable for preventing further accumulation of biogenic amines during the storage of dry fermented sausages.

β-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity. PMID:24710170

Introduction Recent studies reported that human IgG antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2-overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). The objective of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both a breast cancer cell culture method and an in vivo mouse xenograft tumor model. Methods Trastuzumab antibody was incubated with a panel of human matrix metalloproteinases, and proteolytic cleavage in the lower hinge region was detected using both western blotting and mass spectrometry. Single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate ADCC and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in the mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry. Results scIgG-T retains HER2 antigen binding activity and inhibits HER2-mediated downstream signaling and cell proliferation in vitro when compared with the intact trastuzumab. However, scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry showed reduced immune cell infiltration in tumor tissues treated with scIgG-T when compared with those treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro. Conclusion Trastuzumab can be cleaved by matrix metalloproteinases within the lower hinge. scIgG-T exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results

Local delivery of drugs offers the potential for high local drug concentration while minimizing systemic toxicity, which is often observed with oral dosing. However, local depots are typically administered less frequently and include an initial burst followed by a continuous release. To maximize efficiency of therapy, it is critical to ensure that drug is only released when needed. One of the hallmarks of rheumatoid arthritis, for example, is its variable disease activity consisting of exacerbations of inflammation punctuated by periods of remission. This presents significant challenges for matching localized drug delivery with disease activity. An optimal system would be nontoxic and only release drugs during the period of exacerbation, self-titrating in response to the level of inflammation. We report the development of an injectable self-assembled nanofibrous hydrogel, from a generally recognized as safe material, which is capable of encapsulation and release of agents in response to specific enzymes that are significantly upregulated in a diseased state including matrix metalloproteinases (MMP-2 and MMP-9) and esterases. We show that these self-assembled nanofibrous gels can withstand shear forces that may be experienced in dynamic environments such as joints, can remain stable following injection into healthy joints of mice, and can disassemble in vitro to release encapsulated agents in response to synovial fluid from arthritic patients. This novel approach represents a next-generation therapeutic strategy for localized treatment of proteolytic diseases.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo. PMID:26308941

Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

There exists a general recognition of the fact that post translational modification of CYLD protein through proteolytic cleavage by MALT-1 results in sustained cellular NF-kB activity which is conspicuously found to be associated with cancer in general and hematological malignancies in particular. The present study was directed to understand the contribution of MALT-1 and deubiquitinase CYLD to the initiation of T-cell acute lymphoblastic leukemia (T-ALL). Such a study revealed for the first time that the 35kDa CYLD cleaved factor generated by MALT-1 mediated proteolytic cleavage was conspicuously present in human T- ALL subjects of pediatric age group. Further, over-expression of this 35kDa CYLD factor within normal human peripheral blood mononuclear cells had the inherent capacity to program the genome of these cells resulting in T-cell lineage ALL. Based upon these results, we propose that MALT1 inhibitors may be of crucial importance in the treatment of T-ALL subjects of pediatric age group.

ABSTRACT Whether fibroblasts regulate immune response is a crucial issue in the modulation of inflammatory responses. Herein, we demonstrate that foreskin fibroblasts (FFs) potently inhibit CD3+ T cell proliferation through a mechanism involving early apoptosis of activated T cells. Using blocking antibodies, we demonstrate that the inhibition of T cell proliferation occurs through cell-to-cell interactions implicating PD-1 receptor expressed on T cells and its ligands, PD-L1 and PD-L2, on fibroblasts. Dual PD-1 ligand neutralization is required to abrogate (i) binding of the PD-1-Fc fusion protein, (ii) early apoptosis of T cells, and (iii) inhibition of T cell proliferation. Of utmost importance, we provide the first evidence that PD-1 ligand expression is regulated through proteolytic cleavage by endogenous matrix metalloproteinases (MMPs) without transcriptional alteration during culture-time. Using (i) different purified enzymatic activities, (ii) MMP-specific inhibitors, and (iii) recombinant human MMP-9 and MMP-13, we demonstrated that in contrast to CD80/CD86, PD-L1 was selectively cleaved by MMP-13, while PD-L2 was sensitive to broader MMP activities. Their cleavage by exogenous MMP-9 and MMP-13 with loss of PD-1 binding domain resulted in the reversion of apoptotic signals on mitogen-activated CD3+ T cells. We suggest that MMP-dependent cleavage of PD-1 ligands on fibroblasts may limit their immunosuppressive capacity and thus contribute to the exacerbation of inflammation in tissues. In contrast, carcinoma-associated fibroblasts appear PD-1 ligand-depleted through MMP activity that may impair physical deletion of exhausted defective memory T cells through apoptosis and facilitate their regulatory functions. These observations should be considered when using the powerful PD-1/PD-L1 blocking immunotherapies. PMID:27141350

Background A number of studies have shown that peptidases and in particular cysteine peptidases constitute major pathogenicity factors in Entamoeba histolytica. Recent studies have suggested that a considerable number of genes coding for proteolytic enzymes are present within the E. histolytica genome and questions remain about the mode of expression of the various molecules. Results By homology search within the recently published amoeba genome, we identified a total of 86 E. histolytica genes coding for putative peptidases, including 46 recently described peptidase genes. In total these comprise (i) 50 cysteine peptidases of different families but most of which belong to the C1 papain superfamily, (ii) 22 different metallo peptidases from at least 11 different families, (iii) 10 serine peptidases belonging to 3 different families, and (iv) 4 aspartic peptidases of only one family. Using an oligonucleotide microarray, peptidase gene expression patterns of 7 different E. histolytica isolates as well as of heat stressed cells were analysed. A total of 21 out of 79 amoeba peptidase genes analysed were found to be significantly expressed under standard axenic culture conditions whereas the remaining are not expressed or at very low levels only. In heat-stressed cells the expression of 2 and 3 peptidase genes, respectively, were either decreased or increased. Only minor differences were observed between the various isolates investigated, despite the fact that these isolates were originated from asymptomatic individuals or from patients with various forms of amoebic diseases. Conclusion Entamoeba histolytica possesses a large number of genes coding for proteolytic enzymes. Under standard culture conditions or upon heat-stress only a relatively small number of these genes is significantly expressed and only very few variations become apparent between various clinical E. histolytica isolates, calling into question the importance of these enzymes in E. histolytica

The proteolytic processing of the amyloid-beta protein precursor plays a key role in the development of Alzheimer's disease. Cleavage of the amyloid-beta protein precursor may occur via two pathways, both of which involve the action of proteases called secretases. One pathway, involving beta- and gamma-secretase, liberates amyloid-beta protein, a protein associated with the neurodegeneration seen in Alzheimer's disease. The alternative pathway, involving alpha-secretase, precludes amyloid-beta protein formation. In this review, we describe the progress that has been made in identifying the secretases and their potential as therapeutic targets in the treatment or prevention of Alzheimer's disease.

We have created a novel enzyme reactor using electric field-mediated orientation and immobilization of proteolytic enzymes (trypsin/chymotrypsin) on biocompatible PVDF membranes in a continuous flow-through chamber. Using less than 5 minutes, this reactor in various enzyme combinations can produce enhanced rapid digestion for standardized prototypic proteins, hydrophilic proteins and hydrophobic transmembrane proteins when compared to in-solution techniques. With improved digestive efficiency, our reactor improved the overall functional analysis of lipid raft proteomes by identifying more closely functionally linked proteins and elucidated a richer set of biological processes and pathways linked to the proteins than traditional in-solution methods. PMID:21338726

A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis.

Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH/sub 2/-coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X.

The aim of this work was to study the microbiological, physico-chemical and proteolytic changes in Valdeón blue-veined cheese during ripening. Eight replicas of cheese were produced and a total of 48 cheeses were analysed. Lactic acid bacteria, mainly lactococci, were the predominant flora during the early stages of ripening, gradually being replaced by moulds and yeasts (8 log units). Enterococci and Enterobacteriaceae counts were very low or zero. This variety was characterised by a total solids content of 61.80g per 100g(-1) of cheese, a salt/moisture ratio of 8.92g salt per 100g(-1) moisture, a pH of 6.4-7.6 and a water activity of 0.917. At the end of ripening, primary and secondary proteolysis were very high, which resulted in an almost total degradation of αs1- and β-casein (approximately 90%). The peptide profile of the aqueous soluble extracts at pH 4.6 showed great complexity during ripening.

Maturation of the arenavirus GP precursor (GPC) involves proteolytic processing by cellular signal peptidase and the proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), yielding a tripartite complex comprised of a stable signal peptide (SSP), the receptor-binding GP1, and the fusion-active transmembrane GP2. Here we investigated the roles of SKI-1/S1P processing and SSP in the biosynthesis of the recombinant GP ectodomains of lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV). When expressed in mammalian cells, the LCMV and LASV GP ectodomains underwent processing by SKI-1/S1P, followed by dissociation of GP1 from GP2. The GP2 ectodomain spontaneously formed trimers as revealed by chemical cross-linking. The endogenous SSP, known to be crucial for maturation and transport of full-length arenavirus GPC was dispensable for processing and secretion of the soluble GP ectodomain, suggesting a specific role of SSP in the stable prefusion conformation and transport of full-length GPC.

The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, β-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5–7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha–1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies. PMID:20463814

Two ecotypes of Arabidopsis thaliana were transformed with the gene encoding tobacco mosaic virus (TMV) movement protein (P30). P30 accumulated largely in a subcellular fraction containing cell wall components and as a soluble protein. The protein migrated in denaturing gels with an M(r) of 30K, significantly faster than P30 (M(r) approximately 34K) accumulating after expression in transgenic tobacco, Escherichia coli or Spodoptera frugiperda cells, or after virus multiplication in tobacco. The P30 from A. thaliana infected with TMV for 14 days comigrated with that from E. coli, but that from A. thaliana infected for 49 days was of the smaller size. The use of antisera specific for the N- or C-termini of P30 showed that in A. thaliana P30 was proteolytically processed at the N-terminus, a region essential for P30 function. The failure of these plants to complement a TMV P30 mutant indicated that processed P30 was nonfunctional, although the processing was not so rapid that it prevented the development of systemic infections with wild type TMV. The absence of detectable P30 phosphorylation in A. thaliana demonstrated that phosphorylation was not essential for movement protein function and suggested that this species may use proteolytic cleavage of the N-terminus as an alternative strategy to tobacco for deactivating P30.

Degradation and destruction of articular cartilage is the etiology of osteoarthritis (OA), an entity second only to cardiovascular disease as a cause of disability in the United States. Joint mechanics and cartilage biochemistry are believed to play a role in OA; an optical tool to detect structural and chemical changes in articular cartilage might offer benefit for its early detection and treatment. The objective of the present study was to identify the spectral changes in intrinsic ultraviolet (UV) fluorescence of cartilage that occur after proteolytic digestion of cartilage. Bovine articular cartilage samples were incubated in varying concentrations of collagenase ranging from 10ug/mL up to 5mg/mL for 18 hours at 37°C, a model of OA. Pre- and post-incubation measurements were taken of the UV excitation-emission spectrum of each cartilage sample. Mechanical tests were performed to determine the pre- and post-digestion force/displacement ratio associated with indentation of each sample. Spectral changes in intrinsic cartilage fluorescence and stiffness of the cartilage were associated with proteolytic digestion. In particular, changes in the relative intensity of fluorescence peaks associated with pentosidine crosslinks (330 nm excitation, 390 nm emission) and tryptophan (290 nm excitation, 340 nm emission) were found to correlate with different degrees of cartilage digestion and cartilage stiffness. In principle, it may be possible to use UV fluorescence spectral data for early detection of damage to articular cartilage, and as a surrogate measure for cartilage stiffness.

The process of cellular senescence generates a repressive chromatin environment, however, the role of histone variants and histone proteolytic cleavage in senescence remains unclear. Using models of oncogene-induced and replicative senescence, here we report novel histone H3 tail cleavage events mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and the histone variant H3.3 is the preferred cleaved form of H3. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first twenty-one amino acids of the H3 tail, is sufficient to induce senescence. Further, H3.3cs1 chromatin incorporation is mediated by the HUCA histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our study identifies histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence. PMID:25394905

Infusion of cystamine into the isolated, perfused rat liver resulted in tissue damage preceded by the formation of cystamine-protein mixed disulfides which were mainly detected in the plasma membrane fraction. Hepatotoxicity was prevented when dithiothreitol was infused after cystamine or when the calcium antagonist, verapamil, was co-infused with the disulfide. In isolated hepatocytes, the formation of cystamine-protein mixed disulfides was associated with an inhibition of plasma membrane Ca/sup 2 +/-ATPase activity and a decreased rate of Ca/sup 2 +/ efflux from the cells. This resulted in intracellular Ca/sup 2 +/ accumulation which was followed by a stimulation of both phospholipid hydrolysis and proteolysis, as indicated by enhanced rates of release of radioactivity from hepatocytes prelabeled with (/sup 14/C)arachidonate and (/sup 14/C)valine, respectively. Preincubation of hepatocytes with the calmodulin inhibitor, calmidazolium, or with the phospholipase inhibitors, chlorpromazine and dibucaine, inhibited the stimulation of (/sup 14/C)arachidonate release by cystamine. However, none of these agents prevented the onset of cystamine toxicity in hepatocytes. In contrast, pretreatment of the cells with antipain or leupeptin, two inhibitors of Ca/sup 2 +/-activated proteases, abolished the stimulation of proteolysis by cystamine and also protected the cells from cystamine toxicity. Our results suggest that the perturbation of intracellular Ca/sup 2 +/ homeostasis by cystamine is caused by the inhibition of Ca/sup 2 +/ efflux associated with the formation of cystamine-protein mixed disulfides in the plasma membrane and that subsequent cytotoxicity results from Ca/sup 2 +/-activation of a nonlysosomal proteolytic system.

The conserved Notch signaling pathway plays crucial roles in developing and self-renewing tissues. Notch is activated upon ligand-induced conformation change of the Notch negative regulatory region (NRR) unmasking a key proteolytic site (S2) and facilitating downstream events. Thus far, the molecular mechanism of this signal activation is not defined. However, strong indirect evidence favors a model whereby transendocytosis of the Notch extracellular domain, in tight association with ligand into the ligand-bearing cell, exerts a force on the NRR to drive the required structure change. Here, we demonstrate that force applied to the human Notch2 NRR can indeed expose the S2 site and, crucially, allow cleavage by the metalloprotease TACE (TNF-alpha-converting enzyme). Molecular insight into this process is achieved using atomic force microscopy and molecular dynamics simulations on the human Notch2 NRR. The data show near-sequential unfolding of its constituent LNR (Lin12-Notch repeat) and HD (heterodimerization) domains, at forces similar to those observed for other protein domains with a load-bearing role. Exposure of the S2 site is the first force "barrier" on the unfolding pathway, occurring prior to unfolding of any domain, and achieved via removal of the LNRAB linker region from the HD domain. Metal ions increase the resistance of the Notch2 NRR to forced unfolding, their removal clearly facilitating unfolding at lower forces. The results provide direct demonstration of force-mediated exposure and cleavage of the Notch S2 site and thus firmly establish the feasibility of a mechanotransduction mechanism for ligand-induced Notch activation.

Given the importance of protease's worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM). We also quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0.072 and 0.494 mg·mL(-1) of azocasein, resp.) and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%), which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise.

Harvesting and storing high-quality forage in the cool humid regions of agricultural production remains a challenge due to the potentially high degree of protein degradation during ensiling. Red clover is an exception with high protein levels at harvest that are maintained during ensiling. Decreased...

Given the importance of protease's worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM). We also quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0.072 and 0.494 mg·mL−1 of azocasein, resp.) and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%), which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise. PMID:26925415

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, contains two immunodominant proteins, rOmpA and rOmpB, in the outer membrane. Both rOmpA and rOmpB are conserved throughout spotted fever group rickettsiae as members of a family of autotransporter proteins. Previously, it was demonstrated that rOmpB is proteolytically processed, with the cleavage site residing near the autotransporter domain at the carboxy-terminal end of the protein, cleaving the 168-kDa precursor into apparent 120-kDa and 32-kDa fragments. The 120- and 32-kDa fragments remain noncovalently associated on the surface of the bacterium, with implications that the 32-kDa fragment functions as the membrane anchor domain. Here we present evidence for a similar posttranslational processing of rOmpA. rOmpA is expressed as a predicted 224-kDa precursor yet is observed on SDS-PAGE as a 190-kDa protein. A small rOmpA fragment of ∼32 kDa was discovered during surface proteome analysis and identified as the carboxy-terminal end of the protein. A rabbit polyclonal antibody was generated to the autotransporter region of rOmpA and confirmed a 32-kDa fragment corresponding to the calculated mass of a proteolytically cleaved rOmpA autotransporter region. N-terminal amino acid sequencing revealed a cleavage site on the carboxy-terminal side of Ser-1958 in rOmpA. An avirulent strain of R. rickettsii Iowa deficient in rOmpB processing was also defective in the processing of rOmpA. The similarities of the cleavage sites and the failure of R. rickettsii Iowa to process either rOmpA or rOmpB suggest that a single enzyme may be responsible for both processing events.IMPORTANCE Members of the spotted fever group of rickettsiae, including R. rickettsii, the etiologic agent of Rocky Mountain spotted fever, express at least four autotransporter proteins that are protective antigens or putative virulence determinants. One member of this class of proteins, rOmpB, is proteolytically processed to a

It was observed that during fermentative production of recombinant ovine interferon-tau (r-oIFN-tau) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol. It can be concluded that methanol metabolism along with cell lysis towards the end of fermentation contributes to increased proteolyticactivity and eventual degradation of recombinant protein.

Honey is a sweet and healthy food produced by honeybees (Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolyticactivities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification. PMID:23145107

The study of the N-terminome and the precise identification of proteolytic processing events are key in biology. Dedicated methodologies have been developed as the comprehensive characterization of the N-terminome can hardly be achieved by standard proteomics methods. In this context, we have set up a trimethoxyphenyl phosphonium (TMPP) labeling approach that allows the characterization of both N-terminal and internal digestion peptides in a single experiment. This latter point is a major advantage of our strategy as most N-terminomics methods rely on the enrichment of N-terminal peptides and thus exclude internal peptides.We have implemented a double heavy/light TMPP labeling and an automated data validation workflow that make our doublet N-terminal oriented proteomics (dN-TOP) strategy efficient for high-throughput N-terminome analysis.

Fibrin polymerization is a necessary part of hemostasis but clots can obstruct blood vessels and cause heart attacks and strokes. The polymerization reactions are specific and controlled, involving strong knob-into-hole interactions to convert soluble fibrinogen into insoluble fibrin. It has long been assumed that clots and thrombi are stable structures until proteolytic digestion. On the contrary, using the technique of fluorescence recovery after photobleaching, we demonstrate here that there is turnover of fibrin in an uncrosslinked clot. A peptide representing the knobs involved in fibrin polymerization can compete for the holes and dissolve a preformed fibrin clot, or increase the fraction of soluble oligomers, with striking rearrangements in clot structure. These results imply that in vivo clots or thrombi are more dynamic structures than previously believed that may be remodeled as a result of local environmental conditions, may account for some embolization, and suggest a target for therapeutic intervention.

Cell motility is a fundamental physiological process that regulates cellular fate in healthy and diseased systems. Cells cultured in 3D environments often exhibit biphasic dependence of migration speed with cell adhesion. Much is not understood about this very common behavior. A phenomenological model for 3D single-cell migration that exhibits biphasic behavior and highlights the important role of steric hindrance is developed and studied analytically. Changes in the biphasic behavior in the presence of proteolytic enzymes are investigated. Our methods produce a framework to determine analytic formulae for the mean cell speed, allowing general statements in terms of parameters to be explored, which will be useful when interpreting future experimental results. Our formula for mean cell speed as a function of ligand concentration generalizes and extends previous computational models that have shown good agreement with in vitro experiments. PMID:24348878

Amyloid-β (Aβ) peptide accumulation in the brain is central to the pathogenesis of Alzheimer’s disease (AD). Aβ is produced through proteolytic processing of a transmembrane protein, β-amyloid precursor protein (APP), by β- and γ-secretases. Mounting evidence has demonstrated that alterations in APP cellular trafficking and localization directly impact its processing to Aβ. Members of the low-density lipoprotein receptor family, including LRP, LRP1B, SorLA/LR11, and apoER2, interact with APP and regulate its endocytic trafficking. Additionally, APP trafficking and processing are greatly affected by cellular cholesterol content. In this review, we summarize the current understanding of the roles of lipoprotein receptors and cholesterol in APP trafficking and processing and their implication for AD pathogenesis and therapy. PMID:19041409

In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

Dietary gluten proteins from wheat, rye and barley are the primary triggers for the immuno-pathogenesis of Celiac Sprue, a widespread immune disease of the small intestine. Recent molecular and structural analyses of representative gluten proteins, most notably α- and γ-gliadin proteins from wheat, have improved our understanding of these pathogenic mechanisms. In particular, based on the properties of a 33-mer peptide, generated from α-gliadin under physiological conditions, a link between digestive resistance and inflammatory character of gluten has been proposed. Here we report three lines of investigation in support of this hypothesis. First, biochemical and immunological analysis of deletion mutants of α-2 gliadin confirmed that the DQ2 restricted T cell response to the α-2 gliadin are directed towards the epitopes clustered within the 33-mer. Second, proteolytic analysis of a representative γ-gliadin led to the identification of another multivalent 26-mer peptide that was also resistant to further gastric, pancreatic and intestinal brush border degradation, and was a good substrate of human transglutaminase 2 (TG2). Analogous to the 33-mer, the synthetic 26-mer peptide displayed markedly enhanced T cell antigenicity compared to monovalent control peptides. Finally, in silico analysis of the gluten proteome led to the identification of at least 60 putative peptides that share the common characteristics of the 33-mer and the 26-mer peptides. Together, these results highlight the pivotal role of physiologically generated, proteolytically stable, TG2-reactive, multivalent peptides in the immune response to dietary gluten in Celiac Sprue patients. Prolyl endopeptidase treatment was shown to abolish the antigenicity of both the 33-mer and the 26-mer peptides, and was also predicted to have comparable effects on other proline-rich putatively immunotoxic peptides identified from other polypeptides within the gluten proteome. PMID:16212427

An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P1 and P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin {center_dot} APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

In this study, we adapted a FluoSphere bead-binding assay to study the exposure and release of guinea pig sperm acrosomal components during the course of capacitation and acrosomal exocytosis. Prior to capacitation or the initiation of exocytosis, acrosomal proteins were not accessible to FluoSpheres coated with antibodies against two acrosomal matrix (AM) proteins, AM67 and AM50; during the course of capacitation and ionophore-induced acrosomal exocytosis, however, we detected the transient exposure of the solid-phase AM proteins on the surface of guinea pig sperm using the antibody-coated fluorescent beads. Several different transitional stages leading to complete acrosomal exocytosis were classified, and we propose these represent true, functional intermediates since some of the AM proteins are orthologues of mouse proteins that bind the zona pellucida (ZP) of unfertilized eggs. In addition, we present evidence that implicates acrosin in the proteolytic processing of AM50 during AM disassembly. Thus, we propose that the transitional states of acrosomal exocytosis involve early binding of AM proteins to the ZP (by what visually appear to be "acrosome-intact" sperm), maintenance of ZP binding that coincides with the progressive exposure of AM proteins, and gradual proteolytic disassembly of the AM to allow sperm movement through the ZP. We feel this "transitional states" model provides a more refined view of acrosomal function that supports a move away from the widely held, overly simplistic, and binary "acrosome-reaction" model, and embraces a more dynamic view of acrosomal exocytosis that involves intermediate stages of the secretory process in ZP binding and penetration.

In this study, Antheraea pernyi silk fibroin (Ap-SF) films were incubated with Protease Type XXI from Streptomyces griseus, at 37 degrees C, to investigate the degradation behavior in an in vitro model system. The enzyme-resistant fractions of Ap-SF films and the soluble peptides formed by proteolytic degradation were collected at specified times, from 1 to 17 days, and analyzed by high performance liquid chromatography, differential scanning calorimetry, FT-Raman, and FT-IR spectroscopy. Proteolysis resulted in extensive weight loss and progressive fragmentation of films, especially at long degradation times. A range of soluble peptides was formed by proteolysis. By high performance-size exclusion chromatography it was found that their average molecular weight changed with the time of incubation. The chemical analysis of the enzyme-resistant fraction of Ap-SF films at different times of degradation indicated that the proteolytic attack preferentially occurred in the less ordered Gly rich sequences and that the contribution of the Ala rich crystalline regions to the composition of biodegraded films became progressively larger. Accordingly, DSC and spectroscopic results showed an enhancement of the crystalline character of the biodegraded films. From the behavior of the most important thermal transitions, it was deduced that the alpha-helix domains probably represent the most enzyme-resistant fraction. The in vitro approach used in the present study seems to be a valid tool for studying the rate and mechanism of degradation of Ap-SF films and of other biopolymers of potential biomedical utility.

An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P{sub 1} and P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin-APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

Dietary gluten proteins from wheat, rye, and barley are the primary triggers for the immuno-pathogenesis of Celiac Sprue, a widespread immune disease of the small intestine. Recent molecular and structural analyses of representative gluten proteins, most notably alpha- and gamma-gliadin proteins from wheat, have improved our understanding of these pathogenic mechanisms. In particular, based on the properties of a 33-mer peptide, generated from alpha-gliadin under physiological conditions, a link between digestive resistance and inflammatory character of gluten has been proposed. Here, we report three lines of investigation in support of this hypothesis. First, biochemical and immunological analysis of deletion mutants of alpha-2 gliadin confirmed that the DQ2 restricted T cell response to the alpha-2 gliadin are directed toward the epitopes clustered within the 33-mer. Second, proteolytic analysis of a representative gamma-gliadin led to the identification of another multivalent 26-mer peptide that was also resistant to further gastric, pancreatic and intestinal brush border degradation, and was a good substrate of human transglutaminase 2 (TG2). Analogous to the 33-mer, the synthetic 26-mer peptide displayed markedly enhanced T cell antigenicity compared to monovalent control peptides. Finally, in silico analysis of the gluten proteome led to the identification of at least 60 putative peptides that share the common characteristics of the 33-mer and the 26-mer peptides. Together, these results highlight the pivotal role of physiologically generated, proteolytically stable, TG2-reactive, multivalent peptides in the immune response to dietary gluten in Celiac Sprue patients. Prolyl endopeptidase treatment was shown to abolish the antigenicity of both the 33-mer and the 26-mer peptides, and was also predicted to have comparable effects on other proline-rich putatively immunotoxic peptides identified from other polypeptides within the gluten proteome.

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development. PMID:21695060

Myofibrillar protein turnover is a key component of muscle growth and degeneration, requiring proteolytic enzymes to degrade the skeletal muscle proteins. The objective of this study was to investigate the role of the calpain proteolytic system in muscle growth development using µ-calpain knockout (...

The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure. PMID:27535140

Many diverse clinical isolates of Proteus mirabilis (48 strains), P. penneri (25), P. vulgaris biogroup 2 (48) and P. vulgaris biogroup 3 (21) from man were examined for their ability to produce proteolytic enzymes and the nature and characteristics of the proteases were studied. All the P. penneri isolates, most (94-90%) of the P. mirabilis and P. vulgaris biogroup 2 isolates, but only 71% of the P. vulgaris biogroup 3 isolates, secreted proteolytic enzymes. These were detected most readily at pH 8 with gelatin as substrate. A strong correlation was found between the ability of a strain to form swarming growth and its ability to secrete proteases. Non-swarming isolates invariably appeared to be non-proteolytic. However, some isolates, particularly of P. vulgaris biogroup 3, were non-proteolytic even when they formed swarming growth. Analysis of the secreted enzymes of the different Proteus spp. on polyacrylamide-gelatin gels under various constraints of pH and other factors showed that they were all EDTA-sensitive metalloproteinases. Analysis of the kinetics of production of the proteases revealed the formation of an additional protease of undefined type and function that was cell-associated and formed before the others were secreted. The secreted protease was subsequently modified to two isoforms whose mass (53-46 kDa) varied with the Proteus spp. and the strain. There was no evidence that the secreted proteases of strains of Proteus spp. were of types other than metalloproteinases.

Actinobacillus pleuropneumoniae exotoxins (Apx) are major virulence factors that play important roles in the pathogenesis of pleuropneumonia in swine. A previous study has demonstrated that native ApxI at low concentrations induces apoptosis in primary porcine alveolar macrophages (PAMs) via a caspase-3-dependent pathway. However, the molecular mechanisms underlying ApxI-induced apoptosis remain largely unknown. In this study, it was shown that ApxI treatment in PAMs rapidly induced phosphorylation of both p38 and JNK, members of the mitogen-activated protein kinase family. Application of a selective p38 or JNK inhibitor significantly reduced ApxI-induced apoptosis, indicating the involvement of p38 and JNK pathways in this event. Furthermore, activation of both caspase-8 and -9 were observed in ApxI-stimulated PAMs. Inhibition of caspase-8 and caspase-9 activity significantly protected PAMs from ApxI-induced apoptosis. In addition, Bid activation was also noted in ApxI-treated PAMs, and inhibition of caspase-8 suppressed the activation of Bid and caspase-9, suggesting that ApxI was able to activate the caspases-8-Bid-caspase-9 pathway. Notably, inhibition of p38 or JNK pathway greatly attenuated the activation of caspases-3, -8, and -9. This study is the first to demonstrate that ApxI-induced apoptosis of PAMs involves the activation of p38 and JNK, and engages the extrinsic and intrinsic apoptotic pathways.

The human paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) plays a central role in nuclear factor-κB (NF-κB) signaling as both a protease and scaffolding protein. Knocking out MALT1 leads to impaired NF-κB signaling and failure to mount an effective immune response. However, it is unclear to which degree it is the scaffolding function versus the proteolyticactivity of MALT1 that is essential. Previous work involving a MALT1 inhibitor with low selectivity suggests that the enzymatic function plays an important role in different cell lines. To help elucidate this proteolytic role of MALT1, we have designed activity-based probes that inhibit its proteolyticactivity. The probes selectively label active enzyme and can be used to inhibit MALT1 and trace its activity profile, helping to create a better picture of the significance of the proteolytic function of MALT1.

Tumor-associated trypsin-2 and matrix metalloprotease-9 (MMP-9) are associated with cancer, particularly with invasive squamous cell carcinomas. They require activation for catalytical competence via proteolytic cascades. One cascade is formed by enterokinase, trypsin-2 and MMP-9; enterokinase activates trypsinogen-2 to trypsin-2, which is an efficient proMMP-9 activator. We describe here that oral squamous cell carcinomas express all members of this cascade: MMP-9, trypsin-2 and enterokinase. The expression of enterokinase in a carcinoma cell line not derived from the duodenum was shown here for the first time. Enterokinase directly cleaved proMMP-9 at the Lys65-Ser66 site, but failed to activate it in vitro. We demonstrated by confocal microscopy that MMP-9 and trypsin-2 co-localized in intracellular vesicles of the carcinoma cells. This co-localization of trypsin-2 and MMP-9 resulted in intracellular proMMP-9 processing that represented fully or partially activated MMP-9. However, although both proteases were present also in various bone tumor tissues, MMP-9 and trypsin-2 never co-localized at the cellular level in these tissues. This suggests that the intracellular vesicular co-localization, storage and possible activation of these proteases may be a unique feature for aggressive epithelial tumors, such as squamous cell carcinomas, but not for tumors of mesenchymal origin.

Background Clostridium botulinum is a group of four physiologically and phylogenetically distinct bacteria that produce botulinum neurotoxin. While studies have characterised variability between strains of Group I (proteolytic) C. botulinum, the genetic and physiological variability and relationships between strains within Group II (non-proteolytic) C. botulinum are not well understood. In this study the genome of Group II strain C. botulinum Eklund 17B (NRP) was sequenced and used to construct a whole genome DNA microarray. This was used in a comparative genomic indexing study to compare the relatedness of 43 strains of Group II C. botulinum (14 type B, 24 type E and 5 type F). These results were compared with characteristics determined from physiological tests. Results Whole genome indexing showed that strains of Group II C. botulinum isolated from a wide variety of environments over more than 75 years clustered together indicating the genetic background of Group II C. botulinum is stable. Further analysis showed that strains forming type B or type F toxin are closely related with only toxin cluster genes targets being unique to either type. Strains producing type E toxin formed a separate subset. Carbohydrate fermentation tests supported the observation that type B and F strains form a separate subset to type E strains. All the type F strains and most of type B strains produced acid from amylopectin, amylose and glycogen whereas type E strains did not. However, these two subsets did not differ strongly in minimum growth temperature or maximum NaCl concentration for growth. No relationship was found between tellurite resistance and toxin type despite all the tested type B and type F strains carrying tehB, while the sequence was absent or diverged in all type E strains. Conclusions Although Group II C. botulinum form a tight genetic group, genomic and physiological analysis indicates there are two distinct subsets within this group. All type B strains and type F

Metacaspases are cysteine peptidases that are distantly related to the caspases, for which proteolytic processing is central to their activation. Here, we show that recombinant metacaspase 2 (MCA2) from Trypanosoma brucei has arginine/lysine-specific, Ca(2+)-dependent proteolyticactivity. Autocatalytic processing of MCA2 occurred after Lys55 and Lys268; however, this was shown not to be required for the enzyme to be proteolyticallyactive. The necessity of Ca(2+), but not processing, for MCA2 enzymatic activity clearly distinguishes MCA2 from the caspases and would be consistent with different physiological roles.

The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in alpha helices, about a third in beta-sheet structure, and the remainder in beta turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication.

In the present study the contact irritative--eczematogenic--effect of alkaline protease enzymes (PE) contained in syndet laundry detergents has been investigated. The method employed was the occlusive epicutaneous test (ET) involving the use of increasing serial dilutions of PE and various times of exposure. The tests were made on sympton-free skin, on skin showing premorbid or slight irritative changes, on skin previously acid- or base-treated, in the presence of increased skin permeability and following the skin stripping method. The results obtained in the 912 test series indicate that the PEs tested ("Tenzym prilled" Grindstedvoerkek, and "Maxatase" Gist-Brocases) caused no irritation or other damage to the intact or slightly lesioned skin even in response to close contact lasting several days. When as a result of increased irritation, or for other reasons, the PEs may be assumed to penetrate into the subcorneal layers, they may exert a proteolytic effect, as manifested in the increasing number of positive ETs. In such cases the PE concentrations exceeded by far those employed in the commercial detergents. It is most likely that the skin changes noted in connection with the use of bioactive laundry detergents are due not to the PE content of these detergents, but to other factors.

Microwave-assisted proteolytic digestion methods have evolved into a highly effective approach and serve as an alternative to conventional overnight digestion. This approach typically exploits the unique microwave properties to facilitate the digestion of proteins into their peptides within minutes. Conventional digestion is carried out at 37°C while microwave-assisted digestion requires much higher and sometimes inconsistent temperatures. Thus, this study aims to investigate whether the faster reaction rate is due to the microwave quantum effect or the thermal effect. Quantitative mass spectrometry was used to conduct kinetic analysis of tryptic digestion for several proteins by microwave and conventional heating. The percentages of digestion products relative to internal standards showed no significant difference between microwave and conventional heating conditions at the same digestion temperature. The optimum temperature for tryptic digestion was determined to be 50°C. Furthermore, this study compares the digestion completeness indicators of several proteins under microwave and conventional heating. Again, the values obtained from microwave and conventional heating were similar given identical temperatures. The overall results prove that a nonthermal effect does not exist in microwave-assisted tryptic digestion. Therefore, conventional heating at high temperatures (50°C) can be also used to accelerate digestion reactions.

Many neurodegenerative diseases such as Alzheimer disease and Parkinson disease show similar characteristics. They typically show deposits of protein aggregates, the formation of which is considered important in their pathogenesis. Recently, aggregation-prone proteins have been shown to spread between cells and so may contribute to the pathogenesis of diseases like prion disease. Such a pathogenesis pathway is possibly common to many neurodegenerative diseases. If confirmed, it could allow the development of therapeutic interventions against many such diseases. In Parkinson disease, α-synuclein, a major component of cytosolic protein inclusions named Lewy body, has been shown to be released and taken up by cells, which may facilitate its progressive pathological spreading between cells. Accordingly, inhibition of spreading by targeting extracellular α-synuclein may represent a new therapy against Parkinson disease. Research into the intercellular spreading of extracellular protein aggregations of α-synuclein and its clearance pathway are reviewed here with a focus on the proteolytic clearance pathway as a therapeutic target for the treatment of Parkinson disease. Considering the similar characteristics of aggregation-prone proteins, these clearance systems might allow treatment of other neurodegenerative diseases beyond Parkinson disease. PMID:23154633

The in vivo proteolytic digestion of bacterial inclusion bodies (IBs) and the kinetic analysis of the resulting protein fragments is an interesting approach to investigate the molecular organization of these unconventional protein aggregates. In this work, we describe a set of mathematical instruments useful for such analysis and interpretation of observed data. These methods combine numerical estimation of digestion rate and approximation of its high-order derivatives, modelling of fragmentation events from a mixture of Poisson processes associated with differentiated protein species, differential equations techniques in order to estimate the mixture parameters, an iterative predictor-corrector algorithm for describing the flow diagram along the cascade process, as well as least squares procedures with minimum variance estimates. The models are formulated and compared with data, and successively refined to better match experimental observations. By applying such procedures as well as newer improved algorithms of formerly developed equations, it has been possible to model, for two kinds of bacterially produced aggregation prone recombinant proteins, their cascade digestion process that has revealed intriguing features of the IB-forming polypeptides.

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5-8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP.

Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3. PMID:27731380

Background Protein conformation and protein/protein interaction can be elucidated by solution-phase Hydrogen/Deuterium exchange (sHDX) coupled to high-resolution mass analysis of the digested protein or protein complex. In sHDX experiments mutant proteins are compared to wild-type proteins or a ligand is added to the protein and compared to the wild-type protein (or mutant). The number of deuteriums incorporated into the polypeptides generated from the protease digest of the protein is related to the solvent accessibility of amide protons within the original protein construct. Results In this work, sHDX data was collected on a 14.5 T FT-ICR MS. An algorithm was developed based on combinatorial optimization that predicts deuterium exchange with high spatial resolution based on the sHDX data of overlapping proteolytic fragments. Often the algorithm assigns deuterium exchange with single residue resolution. Conclusions With our new method it is possible to automatically determine deuterium exchange with higher spatial resolution than the level of digested fragments. PMID:20701784

Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3.

Many neurodegenerative diseases such as Alzheimer disease and Parkinson disease show similar characteristics. They typically show deposits of protein aggregates, the formation of which is considered important in their pathogenesis. Recently, aggregation-prone proteins have been shown to spread between cells and so may contribute to the pathogenesis of diseases like prion disease. Such a pathogenesis pathway is possibly common to many neurodegenerative diseases. If confirmed, it could allow the development of therapeutic interventions against many such diseases. In Parkinson disease, α-synuclein, a major component of cytosolic protein inclusions named Lewy body, has been shown to be released and taken up by cells, which may facilitate its progressive pathological spreading between cells. Accordingly, inhibition of spreading by targeting extracellular α-synuclein may represent a new therapy against Parkinson disease. Research into the intercellular spreading of extracellular protein aggregations of α-synuclein and its clearance pathway are reviewed here with a focus on the proteolytic clearance pathway as a therapeutic target for the treatment of Parkinson disease. Considering the similar characteristics of aggregation-prone proteins, these clearance systems might allow treatment of other neurodegenerative diseases beyond Parkinson disease.

The aim of this study was to investigate the influence of proteolytic pork hydrolysate (PPH) on yoghurt production by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Fresh lean pork was cut into pieces and mixed with deionized water and dealt with protease, then the resulting PPH was added to milk to investigate the effects of PPH on yoghurt production. The fermentation time, the viable cell counts, the flavor, free amino acids compounds, and sensory evaluation of yoghurt were evaluated. These results showed that PPH significantly stimulated the growth and acidification of the both bacterial strains. When the content of PPH reached 5% (w/w), the increased acidifying rate occurred, which the fermentation time was one hour less than that of the control, a time saving of up to 20% compared with the control. The viable cell counts, the total free amino acids, and the scores of taste, flavor and overall acceptability in PPH-supplemented yoghurt were higher than the control. Furthermore, the contents of some characteristic flavor compounds including acids, alcohols, aldehydes, ketones and esters were richer than the control. We concluded that the constituents of PPH such as small peptide, vitamins, and minerals together to play the stimulatory roles and result in beneficial effect for the yoghurt starter cultures growth.

The aim of this study was to investigate the influence of proteolytic pork hydrolysate (PPH) on yoghurt production by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Fresh lean pork was cut into pieces and mixed with deionized water and dealt with protease, then the resulting PPH was added to milk to investigate the effects of PPH on yoghurt production. The fermentation time, the viable cell counts, the flavor, free amino acids compounds, and sensory evaluation of yoghurt were evaluated. These results showed that PPH significantly stimulated the growth and acidification of the both bacterial strains. When the content of PPH reached 5% (w/w), the increased acidifying rate occurred, which the fermentation time was one hour less than that of the control, a time saving of up to 20% compared with the control. The viable cell counts, the total free amino acids, and the scores of taste, flavor and overall acceptability in PPH-supplemented yoghurt were higher than the control. Furthermore, the contents of some characteristic flavor compounds including acids, alcohols, aldehydes, ketones and esters were richer than the control. We concluded that the constituents of PPH such as small peptide, vitamins, and minerals together to play the stimulatory roles and result in beneficial effect for the yoghurt starter cultures growth. PMID:27621698

CX3CL1 is a unique chemokine that acts both as a transmembrane endothelial adhesion molecule and, upon proteolytic cleavage, a soluble chemoattractant for circulating leukocytes. The constitutive release of soluble CX3CL1 requires the interaction of its transmembrane species with the integral membrane metalloprotease ADAM10, yet the mechanisms governing this process remain elusive. Using single-particle tracking and subdiffraction imaging, we studied how ADAM10 interacts with CX3CL1. We observed that the majority of cell surface CX3CL1 diffused within restricted confinement regions structured by the cortical actin cytoskeleton. These confinement regions sequestered CX3CL1 from ADAM10, precluding their association. Disruption of the actin cytoskeleton reduced CX3CL1 confinement and increased CX3CL1–ADAM10 interactions, promoting the release of soluble chemokine. Our results demonstrate a novel role for the cytoskeleton in limiting membrane protein proteolysis, thereby regulating both cell surface levels and the release of soluble ligand. PMID:25253723

The regenerative capacity of the Central Nervous System (CNS) is a key factor implicated in the pathogenesis of neurodegenerative diseases. In the present study, the regenerative capacity of the CNS is considered using one of the markers of regeneration, Growth Associated Protein-43 (GAP-43) and its proteolytic fragment GAP-43-3 in the Experimental Autoimmune Encephalomyelitis (EAE) animal model of multiple sclerosis. The EAE on Wistar rats was characterized as an adequate model of multiple sclerosis, with typical clinical (pares and paralysis) and morphological (infiltration of spinal cord and deformation of motoneurons) disorders. Normally about 60% of GAP-43 is cleaved by m-calpain and stays in the form of GAP-43-3. During severe form of EAE up to 85% of GAP-43 can be found cleaved. We speculated that the cleavage of GAP-43 can play a crucial role for regenerative capacity of CNS during EAE development. Thus the distribution of GAP-43 and GAP-43-3 in the spinal cord was analyzed. The manifestation of clinical signs of EAE has been found to be in correlation with the levels of GAP-43 proteolysis both in the homogenate of the spinal cord and on the spinal cord slices. The immunoreactive staining enabled the observation of the accumulation of GAP-43-3 predominantly in microglial cells.

ChanSu(CS), a traditional Chinese medicine, is composed of many chemical compoments. It is isolated from the dried white secretion of the auricular and skin glands of toads, and it has been widely used for treating the heart diseases and other systemic illnesses. However, it is difficult to judge antitumor effect of agents derived from ChanSu and the underlying mechanism of ChanSu inducing cell apoptosis is still unclear. This report was performed to explore the inhibitory effect and mechanism of ChanSu on human lung adenocarcinoma cells (ASTC-a-1). Fluorescence emission spectra and fluorescence resonance energy transfer (FRET) were used to study the caspase-3 activation during the ChanSu-induced human lung adenocarcinoma (ASTC-a-1) cell apoptosis. CCK-8 was used to assay the inhibition of ChanSu on the cell viability. The cells expressing stably with SCAT3 was used to examine if caspase-3 was activated by ChanSu using acceptor photobleaching technique. Our data showed that treatment of ASTC-a-1 cell with ChanSu resulted in the inhibition of viability and induction of apoptosis in a dose-dependent manner and the SCAT3 was almost cleaved 24 h after ChanSu treatment, implying that ChanSu induced cell apoptosis via a caspase-3-dependent death pathway. Our findings extend the knowledge about the cellular signaling mechanisms mediating ChanSu-induced apoptosis.

The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (P<0.05). All these results indicated that the culturing conditions affected the expression of the proteolytic system genes in Lactobacillus bulgaricus at the transcription level.

The amyloid precursor protein (APP) is well known to be involved in the pathophysiology of Alzheimer's disease (AD) via its cleavage product amyloid ß (Aß). However, the physiological role of APP, its various proteolytic products and the amyloid precursor-like proteins 1 and 2 (APLP1/2) are still not fully clarified. Interestingly, it has been shown that learning and memory processes represented by functional and structural changes at synapses are altered in different APP and APLP1/2 mouse mutants. In addition, APP and its fragments are implicated in regulating synaptic strength further reinforcing their modulatory role at the synapse. While APLP2 and APP are functionally redundant, the exclusively CNS expressed APLP1, might have individual roles within the synaptic network. The proteolytic product of non-amyloidogenic APP processing, APPsα, emerged as a neurotrophic peptide that facilitates long-term potentiation (LTP) and restores impairments occurring with age. Interestingly, the newly discovered η-secretase cleavage product, An-α acts in the opposite direction, namely decreasing LTP. In this review we summarize recent findings with emphasis on the physiological role of the APP gene family and its proteolytic products on synaptic function and plasticity, especially during processes of hippocampal LTP. Therefore, we focus on literature that provide electrophysiological data by using different mutant mouse strains either lacking full-length or parts of the APP proteins or that utilized secretase inhibitors as well as secreted APP fragments.

The amyloid precursor protein (APP) is well known to be involved in the pathophysiology of Alzheimer's disease (AD) via its cleavage product amyloid ß (Aß). However, the physiological role of APP, its various proteolytic products and the amyloid precursor-like proteins 1 and 2 (APLP1/2) are still not fully clarified. Interestingly, it has been shown that learning and memory processes represented by functional and structural changes at synapses are altered in different APP and APLP1/2 mouse mutants. In addition, APP and its fragments are implicated in regulating synaptic strength further reinforcing their modulatory role at the synapse. While APLP2 and APP are functionally redundant, the exclusively CNS expressed APLP1, might have individual roles within the synaptic network. The proteolytic product of non-amyloidogenic APP processing, APPsα, emerged as a neurotrophic peptide that facilitates long-term potentiation (LTP) and restores impairments occurring with age. Interestingly, the newly discovered η-secretase cleavage product, An-α acts in the opposite direction, namely decreasing LTP. In this review we summarize recent findings with emphasis on the physiological role of the APP gene family and its proteolytic products on synaptic function and plasticity, especially during processes of hippocampal LTP. Therefore, we focus on literature that provide electrophysiological data by using different mutant mouse strains either lacking full-length or parts of the APP proteins or that utilized secretase inhibitors as well as secreted APP fragments. PMID:28163673

Two strictly anaerobic, proteolytic bacterial strains, designated strain D3RC-2(T) and D3RC-3r, were isolated from a cellulose-degrading mixed culture enriched from yak rumen content. The strains were Gram-stain negative and non-spore-forming with cell sizes of 0.5-0.8 x 0.6-2.0 mum. The temperature range for growth was 24-46 degrees C (optimum 38-39 degrees C) and the pH range was between 5.6 and 8.7 (optimum 7.0-7.3). Both strains used soya peptone, tryptone, l-phenylalanine, l-leucine, l-methionine, l-serine, l-valine, l-threonine and l-histidine as carbon and nitrogen sources, but did not use any of the saccharides tested. The major fermentation products from PY medium were acetate, propionate and iso-butyrate. The DNA G+C contents of strains D3RC-2(T) and D3RC-3r were 41.0+/-0.1 mol% and 41.3+/-0.1 mol% (HPLC), respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains represented a new phyletic sublineage within the family Clostridiaceae, with <93.8 % 16S rRNA gene sequence similarity to recognized species. On the basis of the phenotypic, genotypic and physiological evidence, strains D3RC-2(T) and D3RC-3r are proposed as representing a novel species of a new genus, for which the name Proteiniclasticum ruminis gen. nov., sp. nov. is proposed. The type strain of the type species is D3RC-2(T) (=AS 1.5057(T)=JCM 14817(T)).

Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

Leukemia is among the most aggressive and prevalent human malignant carcinoma. Chemotherapy is the preferred therapeutic strategy; however, recurrence of cancer and non-selective cytotoxicity are the major concerns. Unlike synthetic chemotherapeutic agents, mistletoe ribosome-inactivating protein (RIP) displays anti-tumor function in various types of cancers. However, its effect on leukemia cells is little explored. In this study, we assessed the impact of Viscum articulatum RIP (Articulatin-D) on the survival of acute T-cell leukemia cells and the involved molecular and cellular mechanisms. Cell proliferation assay showed that Articulatin-D suppressed the viability of leukemia cells selectively. We further confirmed that the elevation of mitochondrial membrane potential and exposure of phosphatidylserine are the early events of apoptosis induction in Articulatin-D-treated Jurkat cells. Subsequently, we found that Articulatin-D treatment induces apoptosis in Jurkat cells in a time- and concentration-dependent manner. In conclusion, we provided evidence that Articulatin-D efficiently activates caspase-8 involved in extrinsic pathway of apoptosis induction, which ultimately results in caspase-3-dependent DNA fragmentation of Jurkat cells. Further evaluation of Articulatin-D in cell culture and animal models may provide novel information on selective cytotoxicity to acute T-cell leukemia and its involvement in targeting tumor cell survival pathways.